Novel 38594, 57312, 53659, 57250, 63760, 49938, 32146, 57259, 67118, 67067, 62092, FBH58295FL, 57255, and 57255alt molecules and uses therefor

ABSTRACT

The invention provides isolated nucleic acids molecules, designated 38594, 57312, 53659, 57250, 63760, 49938, 32146, 57259, 67118, 67067, 62092, FBH58295FL, 57255, and 57255alt nucleic acid molecules, which encode transporter molecules, including sugar transporters, organic anion transporters, amino acid transporters, and phospholipid transporters. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing 38594, 57312, 53659, 57250, 63760, 49938, 32146, 57259, 67118, 67067, 62092, FBH58295FL, 57255, and 57255alt nucleic acid molecules, host cells into which the expression vectors have been introduced, and non-human transgenic animals in which a 38594, 57312, 53659, 57250, 63760, 49938, 32146, 57259, 67118, 67067, 62092, FBH58295FL, 57255, and 57255alt gene has been introduced or disrupted. The invention still further provides isolated 38594, 57312, 53659, 57250, 63760, 49938, 32146, 57259, 67118, 67067, 62092, FBH58295FL, 57255, and 57255alt polypeptides, fusion polypeptides, antigenic peptides and anti-38594, 57312, 53659, 57250, 63760, 49938, 32146, 57259, 67118, 67067, 62092, FBH58295FL, 57255, and 57255alt antibodies. Diagnostic and therapeutic methods utilizing compositions of the invention are also provided.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/154,419, filed May 22, 2002 (pending), published as U.S. patentapplication Publication No. 2003-0143675 A1 on Jul. 31, 2003, which is:

-   -   a continuation-in-part of U.S. patent application Ser. No.        09/858,194, filed May 14, 2001, published as U.S. patent        application Publication No. 2002-0061590 A1 (abandoned), which        claims the benefit of U.S. Provisional Application Ser. No.        60/204,211, filed May 12, 2000;    -   also a continuation-in-part of U.S. patent application Ser. No.        09/895,811, filed Jun. 29, 2001 (abandoned), which claims the        benefit of U.S. Provisional Application Ser. No. 60/215,376,        filed Jun. 29, 2000;    -   also a continuation-in-part of U.S. patent application Ser. No.        09/919,781, filed Jul. 31, 2001, published as U.S. patent        application Publication No. 2002-0123094 A1 (abandoned), which        claims the benefit of U.S. Provisional Application Ser. No.        60/221,769, filed Jul. 31, 2000;    -   also a continuation-in-part of U.S. patent application Ser. No.        09/957,664, filed Sep. 19, 2001, published as U.S. patent        application Publication No. 2002-0123097 A1 (abandoned), which        claims the benefit of U.S. Provisional Application Ser. No.        60/233,790, filed Sep. 19, 2000;    -   also a continuation-in-part of U.S. patent application Ser. No.        09/964,295, filed Sep. 25, 2001, published as U.S. patent        application Publication No. 2003-0050441 A1 (abandoned), which        claims the benefit of U.S. Provisional Application Ser. No.        60/235,107, filed Sep. 25, 2000;    -   also a continuation-in-part of U.S. patent application Ser. No.        09/972,724, filed Oct. 5, 2001, published as U.S. patent        application Publication No. 2002-0103351 A1 (pending), which        claims the benefit of U.S. Provisional Application Ser. No.        60/238,336, filed Oct. 5, 2000;    -   also a continuation-in-part of U.S. patent application Ser. No.        10/002,769, filed Nov. 14, 2001, published as U.S. patent        application Publication No. 2002-0132298 A1 (abandoned), which        claims the benefit of U.S. Provisional Application Ser. No.        60/248,364, filed Nov. 14, 2000, and U.S. Provisional        Application Ser. No. 60/248,878, filed Nov. 15, 2000;    -   also a continuation-in-part of U.S. patent application Ser. No.        10/024,623, filed Dec. 17, 2001, published as U.S. patent        application Publication No. 2002-0187524 A1 (pending), which        claims the benefit of U.S. Provisional Application Ser. No.        60/256,240, filed Dec. 15, 2000, U.S. Provisional Application        Ser. No. 60/256,588, filed Dec. 18, 2000, and U.S. Provisional        Application Ser. No. 60/258,028, filed Dec. 21, 2000;    -   also a continuation-in-part of U.S. patent application Ser. No.        10/055,025, filed Jan. 22, 2002, published as U.S. patent        application Publication No. 2002-0177148 A1 (abandoned), which        claims the benefit of U.S. Provisional Application Ser. No.        60/263,169, filed Jan. 22, 2001; and    -   also claims the benefit of U.S. Provisional Application Ser. No.        60/324,016, filed Sep. 20, 2001 (abandoned).

The entire contents of each of the above-referenced patent applicationsare incorporated herein by this reference.

BACKGROUND OF THE INVENTION

Transport of larger molecules takes place by the action of ‘permeases’and ‘transporters’, two other classes of membrane-localized proteinswhich serve to move charged molecules from one side of a cellularmembrane to the other. Unlike channel molecules, which permitdiffusion-limited solute movement of a particular solute, these proteinsrequire an energetic input, either in the form of a diffusion gradient(permeases) or through coupling to hydrolysis of an energetic molecule(e.g., ATP or GTP) (transporters). The permeases, integral membraneproteins often having between 6-14 membrane-spanning α-helices) enablethe facilitated diffusion of molecules such as glucose or other sugarsinto the cell when the concentration of these molecules on one side ofthe membrane is greater than that on the other. Permeases do not formopen channels through the membrane, but rather bind to the targetmolecule at the surface of the membrane and then undergo aconformational shift such that the target molecule is released on theopposite side of the membrane.

Transport molecules are specific for a particular target solute or classof solutes, and are also present in one or more specific membranes.Transport molecules localized to the plasma membrane permit an exchangeof solutes with the surrounding environment, while transport moleculeslocalized to intracellular membranes (e.g., membranes of themitochondrion, peroxisome, lysosome, endoplasmic reticulum, nucleus, orvacuole) permit import and export of molecules from organelle toorganelle or to the cytoplasm. For example, in the case of themitochondrion, transporters in the inner and outer mitochondrialmembranes permit the import of sugar molecules, calcium ions, and water(among other molecules) into the organelle and the export of newlysynthesized ATP to the cytosol.

Membrane transport molecules (e.g., channels/pores, permeases, andtransporters) play important roles in the ability of the cell toregulate homeostasis, to grow and divide, and to communicate with othercells, e.g., to secrete and receive signaling molecules, such ashormones, reactive oxygen species, ions, neurotransmitters, andcytokines. A wide variety of human diseases and disorders are associatedwith defects in transporter or other membrane transport molecules,including certain types of liver disorders (e.g., due to defects intransport of long-chain fatty acids (Al Odaib et al. (1998) New Eng. J.Med. 339: 1752-1757)), hyperlysinemia (due to a transport defect oflysine into mitochondria (Oyanagi et al. (1986) Inherit. Metab. Dis. 9:313-316), and cataract (Wintour (1997) Clin. Exp. Pharmacol. Physiol24(1):1-9).

Organic anion transporters are a particular subclass of transporterswhich are specific for the transport of organic anions, which include awide variety of drugs and xenobiotics, many of which are harmful to thebody. In addition, organic ion transporters are responsible for thetransport of the metabolites of most lipophilic compounds, e.g., sulfateand glucuronide conjugates (Moller, J. V. and Sheikh, M. I. (1982)Pharmacol. Rev. 34:315-358; Pritchard, J. B. and Miller, D. S. (1993)Physiol. Rev. 73:765-796; Ullrich, K. J. (1997) J. Membr. Biol.158:95-107; Ullrich, K. J. and Rumrich, G. (1993) Clin. Investig.71:843-848; Petzinger, E. (1994) Rev. Physiol. Biochem. Pharmacol.123:47-211).

Sugar transporters are members of the major facilitator superfamily oftransporters. These transporters are passive in the sense that they aredriven by the substrate concentration gradient and they exhibit distinctkinetics as well as sugar substrate specificity. Members of this familyshare several characteristics: (1) they contain twelve transmembranedomains separated by hydrophilic loops; (2) they have intracellular N-and C-termini; and (3) they are thought to function as oscillatingpores. The transport mechanism occurs via sugar binding to the exofacialbinding site of the transporter, which is thought to trigger aconformational change causing the sugar binding site to re-orient to theendofacial conformation, allowing the release of substrate. Thesetransporters are specific for various sugars and are found in bothprokaryotes and eukaryotes. In mammals, sugar transporters transportvarious monosaccharides across the cell membrane (Walmsley et al. (1998)Trends in Biochem. Sci. 23:476-481; Barrett et al. (1999) Curr. Op. CellBiol. 11:496-502).

At least nine mammalian glucose transporters have been identified,GLUT1-GLUT9, which are expressed in a tissue-specific manner (e.g., inbrain, erythrocyte, kidney, muscle, and adipose tissues) (Shepherd etal. (1999) N. Engl. J. Med. 341:248-257; Doege et al. (2000) Biochem. J.350:771-776). Some GLUT proteins have been shown to be present in lowamounts at the plasma membrane during the basal state, at which timelarge amounts are sequestered in intracellular vesicle stores.Stimulatory molecules specific for each GLUT (such as insulin) regulatethe translocation of the GLUT-containing vesicles to the plasmamembrane. The vesicles fuse at the membrane and subsequently expose theGLUT protein to the extracellular milieu to allow glucose (and othermonosaccharide) transport into the cell (Walmsley et al. (1998) Trendsin Biochem. Sci. 23:476-481; Barrett et al. (1999) Curr. Op. Cell Biol.11:496-502). Other GLUT transporters play a role in constitutive sugartransport.

The E1-E2 ATPase family is a large superfamily of transport enzymes thatcontains at least 80 members found in diverse organisms such asbacteria, archaea, and eukaryotes (Palmgren, M. G. and Axelsen, K. B.(1998) Biochim. Biophys. Acta. 1365:37-45). These enzymes are involvedin ATP hydrolysis-dependent transmembrane movement of a variety ofinorganic cations (e.g., H⁺, Na⁺, K⁺, Ca²⁺, Cu²⁺, Cd⁺, and Mg²⁺ ions)across a concentration gradient, whereby the enzyme converts the freeenergy of ATP hydrolysis into electrochemical ion gradients. E1-E2ATPases are also known as “P-type” ATPases, referring to the existenceof a covalent high-energy phosphoryl-enzyme intermediate in the chemicalreaction pathway of these transporters. Until recently, the superfamilycontained four major groups: Ca²⁺ transporting ATPases; Na⁺/K⁺— andgastric H⁺/K⁺ transporting ATPases; plasma membrane H⁺ transportingATPases of plants, fungi, and lower eukaryotes; and all bacterial P-typeATPases (Kuhlbrandt et al. (1998) Curr. Opin. Struct. Biol. 8:510-516).

E1-E2 ATPases are phosphorylated at a highly conserved DKTG sequence.Phosphorylation at this site is thought to control the enzyme'ssubstrate affinity. Most E1-E2 ATPases contain ten alpha-helicaltransmembrane domains, although additional domains may be present. Amajority of known gated-pore translocators contain twelve alpha-helices,including Na⁺/H⁺ antiporters (West (1997) Biochim. Biophys. Acta1331:213-234).

Members of the E1-E2 ATPase superfamily are able to generateelectrochemical ion gradients which enable a variety of processes in thecell such as absorption, secretion, transmembrane signaling, nerveimpulse transmission, excitation/contraction coupling, and growth anddifferentiation (Scarborough (1999) Curr. Opin. Cell Biol. 11:517-522).These molecules are thus critical to normal cell function and well-beingof the organism.

Recently, a new class of E1-E2 ATPases was identified, theaminophospholipid transporters or translocators. These transporterstransport not cations, but phospholipids (Tang, X. et al. (1996) Science272:1495-1497; Bull, L. N. et al. (1998) Nat. Genet. 18:219-224; Mauro,I. et al. (1999) Biochem. Biophys. Res. Commun. 257:333-339). Thesetransporters are involved in cellular functions including bile acidsecretion and maintenance of the asymmetrical integrity of the plasmamembrane.

The histidine triad (HIT) family of proteins are a superfamily ofnucleotide-binding proteins which were first identified based onsequence similarity. Specifically, HIT proteins all have the histidinetriad-containing sequence motif His-φ-His-φ-His-φ-φ, where φ representsa hydrophobic amino acid residue (Seraphin, B. (1992) DNA Sequence3:177-179). The histidine triad motif is responsible for the nucleotidebinding properties of the HIT proteins (Brenner, C. et al. (1999) J.Cell. Physiol. 181:19-187).

The HIT family can be divided into two branches, the Fhit branch and theHint branch. Fhit proteins are found only in animals and fungi, whileHint proteins are found in all forms of cellular life (Brenner et al.(1999) supra). Hint proteins, first purified from rabbit heart cytosol(Gilmour et al. (1997)), are intracellular receptors for purinemononucleotides.

Fhit proteins bind and cleave diadenosine polyphosphates (Ap_(n)A) suchas ApppA and AppppA (Brenner et al. (1999) supra). Human Fhit is a tumorsuppressor protein frequently mutated in cancers of the gastrointestinaltract (Ohta, M. et al. (1996) Cell 84:587-597), lung (Sozzi, G. et al.(1996) Cell 85:17-26), and other tissues.

Under the current model, cellular stress signals cause tRNA synthetasesto produce Ap_(n)A rather than deliver amino acids to tRNAs (Brenner etal. (1999) supra). Fhit acts as a sensor for Ap_(n)A, and Fhit-Ap_(n)Acomplexes stimulate the pro-apoptotic activity of nitrilases, enzymeswhich convert nitriles (such as indoleacetonitrile) to the correspondingacids (such as indoleacetic acid) plus ammonia by addition of two watermolecules. When Fhit is mutated cells cannot sense Ap_(n)A stresssignals, which can result in uncontrolled growth.

Given the important biological and physiological roles played by theE1-E2 ATPase family of proteins and the HIT family of proteins, thereexists a need to identify novel E1-E2 ATPase and HMT family members foruse in a variety of diagnostic/prognostic as well as therapeuticapplications.

The uptake of amino acids in mammalian cells is mediated byenergy-dependent and passive amino acid transporters with different butoverlapping specificities. Different cells contain a distinct set oftransport systems in their plasma membranes. Most energy-dependenttransporters are coupled to the countertransport of K⁺ or to thecotransport of Na⁺ or Cl⁻. Passive transporters are either facilitatedtransporters or channels. The transport of amino acids is important insuch functions as protein synthesis, hormone metabolism, nervetransmission, cellular activation, regulation of cell growth, productionof metabolic energy, synthesis of purines and pyrimidines, nitrogenmetabolism, and/or biosynthesis of urea. Catagna, et al. (1997) TheJournal of Experimental Biology 200:269-286. Examples of important aminoacid transport systems and their physiological roles follow.

L-glutamate is the major mediator of excitatory neurotransmission in themammalian central nervous system. At least four different glutamatetransporters have been cloned, EAAC1, GLT-1, GLAST, and EAAT4. Catagna,et al. (1997) The Journal of Experimental Biology 200:269-286.L-glutamate is stored in synaptic vesicles at presynaptic terminals andreleased into the synaptic cleft to act on glutamate receptors.Glutamate is involved in most aspects of brain function includingcognition, memory, and learning. The role of amino acid transporters inkeeping the extracellular concentration of glutamate low is importantfor the following reasons: (1) to ensure a high signal-to-noise ratioduring neurotransmission; and (2) to prevent neuronal cell deathresulting from excessive activation of glutamate receptors. Glutamatetransporters play a role in stroke, central nervous system ischemia,seizures, and neurodegenerative diseases such as Alzheimer's disease andamyotrophic lateral sclerosis (ALS). Seal (1999) Annu. Rev. Pharmacol.Toxicol. 39:431-56.

A defect in cystine transport during renal cystine reabsorption resultsin cystinuria, an autosomal recessive disorder and a common hereditarycause of nephrolithiasis. The low solubility of cystine in urine favorsformation of cystine-containing kidney stones. At least 2 separate aminoacid transporters are involved in cystine transport: one located in theproximal tubule S1 segment and the other located in the proximal tubuleS3 segment. It is believed that the D2/NBAT amino acid transport systemtransports cystine at the proximal tubule S3 segment.

Cationic amino acid (CAT) transporters are needed for protein synthesis,urea synthesis (arginine), and as precursors of bioactive molecules.Palacin, et al. Physiological Reviews 78(4):969-1054. Arginine is theimmediate precursor for the synthesis of nitric oxide. Nitric oxide actsas a vasodilator where it plays an important role in the regulation ofblood flow and blood pressure. Nitric oxide is also important inneurotransmission. Arginine is also a precursor for the synthesis ofcreatine, which is a high energy phosphate source for musclecontraction. Ornithine is required for the synthesis of polyamines,which are important in cell and tissue growth.

Growth factors, cytokines, and hormones modulate amino acid transport.Kilberg, et al. (1993) Annu. Rev. Nutr. 13:137-65. For example,epidermal growth factor stimulates amino acid transport Systems A and Lin rat kidney cells. Glucagon and glucocorticoid hormones are known tostimulate Systems A and N. Both TNF and IL-1 stimulate SystemASC-mediated glutamine uptake by cultured porcine endothelial cells.Further, TGF-β stimulates both Systems A and L in rat kidney cells.

Given the important role of amino acid transporters in regulating a widevariety of cellular processes, there exists a need for theidentification of novel amino acid transporters as well as modulators ofsuch transporters for use in a variety of pharmaceutical and therapeuticapplications. INDEX Chapter Page Title I. 7 38594, A NOVEL HUMANTRANSPORTER AND USES THEREOF; BRIEF DESCRIPTION OF DRAWINGS II. 17 57312AND 53659, NOVEL HUMAN ORGANIC ANION TRANSPORTER MOLECULES AND USESTHEREOF III. 25 57250, A NOVEL HUMAN SUGAR TRANSPORTER FAMILY MEMBER ANDUSES THEREOF IV. 31 63760, A NOVEL HUMAN TRANSPORTER AND USES THEREOF V.39 49938, A NOVEL HUMAN PHOSPHOLIPID TRANSPORTER AND USES THEREFOR VI.49 32146 AND 57259, NOVEL HUMAN TRANSPORTERS AND USES THEREOF VII. 5867118, 67067, AND 62092, HUMAN PROTEINS AND METHODS OF USE THEREOF VIII.70 FBH58295FL, A NOVEL HUMAN AMINO ACID TRANSPORTER AND USES THEREOF IX.77 57255 and 57255alt, NOVEL HUMAN SUGAR TRANSPORTERS AND USES THEREFORX 83 FURTHER EMBODIMENTS OF 38594, 57312, 53659, 57250, 63760, 49938,32146, 57239, 67118, 67067, 62092, FPH58295FL, 57255 AND 57255altChapter I. 38594, A NOVEL HUMAN TRANSPORTER AND USES THEREOF

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel members of the family of transporter molecules, referred to hereinas MTP-1 nucleic acid and protein molecules. The present invention isalso based, at least in part, on the realization that MTP-1 moleculesare related to ABC transporter molecules, which function in cellulartransmembrane lipid transport, and that MTP-1 molecules arepreferentially expressed in myelo-lymphatic tissue. As such, thefunctioning of MTP-1 molecules may be causatively linked tohematopoietic and immunological diseases, or diseases related to lipidmetabolism, e.g., atherosclerosis. Accordingly, in one aspect, thisinvention provides isolated nucleic acid molecules encoding MTP-1proteins or biologically active portions thereof, as well as nucleicacid fragments suitable as primers or hybridization probes for thedetection of MTP-1-encoding nucleic acids.

In one embodiment, an MTP-1 nucleic acid molecule of the invention is atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or more identical to the nucleotide sequence (e.g., to the entirelength of the nucleotide sequence) shown in SEQ ID NO: 1 or 3, or acomplement thereof.

In a preferred embodiment, the isolated nucleic acid molecule includesthe nucleotide sequence shown in SEQ ID NO:1 or 3, or a complementthereof. In another embodiment, the nucleic acid molecule includes SEQID NO:3 and nucleotides 1-107 of SEQ ID NO:1. In yet a furtherembodiment, the nucleic acid molecule includes SEQ ID NO:3 andnucleotides 1494-1929 of SEQ ID NO:1. In another preferred embodiment,the nucleic acid molecule consists of the nucleotide sequence shown inSEQ ID NO:1 or 3.

In another embodiment, an MTP-1 nucleic acid molecule includes anucleotide sequence encoding a protein having an amino acid sequencesufficiently identical to the amino acid sequence of SEQ ID NO:2. In apreferred embodiment, an MTP-1 nucleic acid molecule includes anucleotide sequence encoding a protein having an amino acid sequence atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or more identical to the entire length of the amino acid sequence ofSEQ ID NO:2.

In another preferred embodiment, an isolated nucleic acid moleculeencodes the amino acid sequence of human MTP-1. In yet another preferredembodiment, the nucleic acid molecule includes a nucleotide sequenceencoding a protein having the amino acid sequence of SEQ ID NO:2. In yetanother preferred embodiment, the nucleic acid molecule is at least50-100, 100-500, 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000,3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000,6000-6500, 6500-6700, or more nucleotides in length. In a furtherpreferred embodiment, the nucleic acid molecule is at least 50-100,100-500, 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000,3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000,6000-6500, 6500-6700, or more nucleotides in length and encodes aprotein having an MTP-1 activity (as described herein).

Another embodiment of the invention features nucleic acid molecules,preferably MTP-1 nucleic acid molecules, which specifically detect MTP-1nucleic acid molecules relative to nucleic acid molecules encodingnon-MTP-1 proteins. For example, in one embodiment, such a nucleic acidmolecule is at least 50-100, 100-500, 500-1000, 1000-1500, 1500-2000,2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000,5000-5500, 5500-6000, 6000-6500, 6500-6700, or more nucleotides inlength and hybridizes under stringent conditions to a nucleic acidmolecule comprising the nucleotide sequence shown in SEQ ID NO: 1, or acomplement thereof.

In preferred embodiments, the nucleic acid molecules are at least 15(e.g., 15 contiguous) nucleotides in length and hybridize understringent conditions to the nucleotide molecules set forth in SEQ ID NO:1.

In other preferred embodiments, the nucleic acid molecule encodes anaturally occurring allelic variant of a polypeptide comprising theamino acid sequence of SEQ ID NO:2, wherein the nucleic acid moleculehybridizes to a nucleic acid molecule comprising SEQ ID NO:1 or 3,respectively, under stringent conditions.

Another embodiment of the invention provides an isolated nucleic acidmolecule which is antisense to an MTP-1 nucleic acid molecule, e.g., thecoding strand of an MTP-1 nucleic acid molecule.

Another aspect of the invention provides a vector comprising an MTP-1nucleic acid molecule. In certain embodiments, the vector is arecombinant expression vector. In another embodiment, the inventionprovides a host cell containing a vector of the invention. In yetanother embodiment, the invention provides a host cell containing anucleic acid molecule of the invention. The invention also provides amethod for producing a protein, preferably an MTP-1 protein, byculturing in a suitable medium, a host cell, e.g., a mammalian host cellsuch as a non-human mammalian cell, of the invention containing arecombinant expression vector, such that the protein is produced.

Another aspect of this invention features isolated or recombinant MTP-1proteins and polypeptides. In one embodiment, an isolated MTP-1 proteinincludes at least one or more of the following domains: a transmembranedomain, and/or an ABC transporter domain.

In a preferred embodiment, an MTP-1 protein includes at least one ormore of the following domains: a transmembrane domain, an ABCtransporter domain, and has an amino acid sequence at least about 50%,55%, 60%, 65%, 67%, 68%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence of SEQ ID NO:2. Inanother preferred embodiment, an MTP-1 protein includes at least one ormore of the following domains: a transmembrane domain, an ABCtransporter domain and has an MTP-1 activity (as described herein).

In yet another preferred embodiment, an MTP-1 protein includes at leastone or more of the following domains: a transmembrane domain, an ABCtransporter domain, and is encoded by a nucleic acid molecule having anucleotide sequence which hybridizes under stringent hybridizationconditions to a nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO: 1 or 3.

In another embodiment, the invention features fragments of the proteinhaving the amino acid sequence of SEQ ID NO:2, wherein the fragmentcomprises at least 15 amino acids (e.g., contiguous amino acids) of theamino acid sequence of SEQ ID NO:2. In another embodiment, an MTP-1protein has the amino acid sequence of SEQ ID NO:2.

In another embodiment, the invention features an MTP-1 protein which isencoded by a nucleic acid molecule consisting of a nucleotide sequenceat least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or more identical to a nucleotide sequence of SEQ ID NO: 1or 3, or a complement thereof. This invention further features an MTP-1protein which is encoded by a nucleic acid molecule consisting of anucleotide sequence which hybridizes under stringent hybridizationconditions to a nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO: 1 or 3, or a complement thereof.

The proteins of the present invention or portions thereof, e.g.,biologically active portions thereof, can be operatively linked to anon-MTP-1 polypeptide (e.g., heterologous amino acid sequences) to formfusion proteins. The invention further features antibodies, such asmonoclonal or polyclonal antibodies, that specifically bind proteins ofthe invention, preferably MTP-1 proteins. In addition, the MTP-1proteins or biologically active portions thereof can be incorporatedinto pharmaceutical compositions, which optionally includepharmaceutically acceptable carriers.

In another aspect, the present invention provides a method for detectingthe presence of an MTP-1 nucleic acid molecule, protein, or polypeptidein a biological sample by contacting the biological sample with an agentcapable of detecting an MTP-1 nucleic acid molecule, protein, orpolypeptide such that the presence of an MTP-1 nucleic acid molecule,protein or polypeptide is detected in the biological sample.

In another aspect, the present invention provides a method for detectingthe presence of MTP-1 activity in a biological sample by contacting thebiological sample with an agent capable of detecting an indicator ofMTP-1 activity such that the presence of MTP-1 activity is detected inthe biological sample.

In another aspect, the invention provides a method for modulating MTP-1activity comprising contacting a cell capable of expressing MTP-1 withan agent that modulates MTP-1 activity such that MTP-1 activity in thecell is modulated. In one embodiment, the agent inhibits MTP-1 activity.In another embodiment, the agent stimulates MTP-1 activity. In oneembodiment, the agent is an antibody that specifically binds to an MTP-1protein. In another embodiment, the agent modulates expression of MTP-1by modulating transcription of an MTP-1 gene or translation of an MTP-1mRNA. In yet another embodiment, the agent is a nucleic acid moleculehaving a nucleotide sequence that is antisense to the coding strand ofan MTP-1 mRNA or an MTP-1 gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant or unwantedMTP-1 protein or nucleic acid expression or activity by administering anagent which is an MTP-1 modulator to the subject. In one embodiment, theMTP-1 modulator is an MTP-1 protein. In another embodiment the MTP-1modulator is an MTP-1 nucleic acid molecule. In yet another embodiment,the MTP-1 modulator is a peptide, peptidomimetic, or other smallmolecule. In a preferred embodiment, the disorder characterized byaberrant or unwanted MTP-1 protein or nucleic acid expression is atransporter-associated disorder.

The present invention also provides diagnostic assays for identifyingthe presence or absence of a genetic alteration characterized by atleast one of (i) aberrant modification or mutation of a gene encoding anMTP-1 protein; (ii) mis-regulation of the gene; and (iii) aberrantpost-translational modification of an MTP-1 protein, wherein a wild-typeform of the gene encodes a protein with an MTP-1 activity.

In another aspect the invention provides methods for identifying acompound that binds to or modulates the activity of an MTP-1 protein, byproviding an indicator composition comprising an MTP-1 protein havingMTP-1 activity, contacting the indicator composition with a testcompound, and determining the effect of the test compound on MTP-1activity in the indicator composition to identify a compound thatmodulates the activity of an MTP-1 protein.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the results of a search which was performed against theMEMSAT database and which resulted in the identification of twelve“transmembrane domains” in the full length human MTP-1 protein (SEQ IDNO:2).

FIGS. 2A-C depict the results of a TaqMan analysis of the relativeexpression of MTP-1 mRNA in a variety of tissues.

FIG. 3 depicts an alignment of the human OAT5 gene with the human OATPegene (GenBank Accession No. AB031051; SEQ ID NO:10). Identical aminoacid residues are indicated by stars.

FIG. 4 depicts a structural, hydrophobicity, and antigenicity analysisof the human OAT4 protein. The locations of the 12 transmembrane domainsare indicated (TM 1, 2, 3, etc.).

FIG. 5 depicts a structural, hydrophobicity, and antigenicity analysisof the human OAT5 protein. The locations of the 12 transmembrane domainsare indicated (TM 1, 2, 3, etc.).

FIG. 6 depicts the expression levels of human OAT5 mRNA in various humancell types and tissues, as determined by Taqman analysis. Samples: (1)normal artery; (2) diseased aorta; (3) normal vein; (4) coronary smoothmuscle cells; (5) human umbilical vein endothelial cells (HUVECs); (6)hemangioma; (7) normal heart; (8) heart—congestive heart failure (CHF);(9) kidney; (10) skeletal muscle; (11) normal adipose tissue; (12)pancreas; (13) primary osteoblasts; (14) differentiated osteoclasts;(15) normal skin; (16) normal spinal cord; (17) normal brain cortex;(18) brain—hypothalamus; (19) nerve; (20) dorsal root ganglion (DRG);(21) normal breast; (22) breast tumor; (23) normal ovary; (24) ovarytumor; (25) normal prostate; (26) prostate tumor; (27) salivary gland;(28) normal colon; (29) colon tumor; (30) normal lung; (31) lung tumor;(32) lung—chronic obstructive pulmonary disease (COPD); (33)colon—inflammatory bowel disease (IBD); (34) normal liver; (35)liver—fibrosis; (36) normal spleen; (37) normal tonsil; (38) normallymph node; (39) normal small intestine; (40) macrophages; (41)synovium; (42) bone marrow mononuclear cells (BM-MNC); (43) activatedperipheral blood mononuclear cells (PBMCs); (44) neutrophils; (45)megakaryocytes; (46) erythroid cells; (47) positive control.

FIG. 7 depicts a structural, hydrophobicity, and antigenicity analysisof the human HST-1 polypeptide.

FIG. 8 depicts the results of a search which was performed against theMEMSAT database and which resulted in the identification of twelve“transmembrane domains” in the human HST-1 polypeptide (SEQ ID NO:13).

FIG. 9 depicts an alignment of the human HST-1 amino acid sequence (SEQID NO: 13) with the amino acid sequence of a human potent brain typeorganic ion transporter (Accession No. AB040056) using the CLUSTAL W(1.74) alignment program.

FIG. 10 is a graph depicting the expression of human HST-1 cDNA (SEQ IDNO:13) in various human tissues as determined by Taqman analysis.

FIG. 11 depicts a structural, hydrophobicity, and antigenicity analysisof the human TP-2 polypeptide.

FIG. 12 depicts the results of a search which was performed against theMEMSAT database and which resulted in the identification of twelve“transmembrane domains” in the human TP-2 polypeptide (SEQ ID NO: 16).

FIG. 13 depicts an alignment of the human TP-2 amino acid sequence (SEQID NO: 16) with the amino acid sequences of the Salmonella typhitetracycline-6-hydroxylase/oxygenase homolog gene (SEQ ID NO: 18) usingthe CLUSTAL W™ (1.74) alignment program.

FIGS. 14A-B depict a Clustal W (1.74) alignment of the human PLTR-1amino acid sequence (“Fbh49938pat”; SEQ ID NO:20) with the amino acidsequence of human FIC1 (“hFIC1_AT1C_”; SEQ ID NO:22). The transmembranedomains (“TM1”, “TM2”, etc.), E1-E2 ATPases phosphorylation site(“phosphorylation site”), and phospholipid transporter specific aminoacid residues (“phospholipid transport”) are boxed.

FIG. 15 depicts a structural, hydrophobicity, and antigenicity analysisof the human PLTR-1 polypeptide. The locations of the 12 transmembranedomains, as well as the E1-E2 ATPase domain, are indicated.

FIG. 16 depicts a structural, hydrophobicity, and antigenicity analysisof the human TFM-2 polypeptide.

FIG. 17 depicts the results of a search which was performed against theMEMSAT database and which resulted in the identification of ten“transmembrane domains” in the human TFM-2 polypeptide (SEQ ID NO:28).

FIG. 18 depicts a structural, hydrophobicity, and antigenicity analysisof the human TFM-3 polypeptide.

FIG. 19 depicts the results of a search which was performed against theMEMSAT database and which resulted in the identification of nine“transmembrane domains” in the human TFM-3 polypeptide (SEQ ID NO:31).

FIG. 20 depicts a structural, hydrophobicity, and antigenicity analysisof the human 67118 polypeptide.

FIGS. 21A-B depict a Clustal W (1.74) alignment of the human 67118 aminoacid sequence (“Fbh67118pat”; SEQ ID NO:34) with the amino acid sequenceof mouse Potential Phospholipid-Transporting ATPase IH (mouseAT1H)(GenBank Accession No. P98197; SEQ ID NO:46). The transmembrane domains(“TM1”, “TM2”, etc.), E1-E2 ATPases phosphorylation site(“phosphorylation site”), and phospholipid transporter specific aminoacid residues (“phospholipid transport”) are boxed.

FIG. 22 depicts a structural, hydrophobicity, and antigenicity analysisof the human 67067 polypeptide.

FIGS. 23A-B depict a Clustal W (1.74) alignment of the human 67067 aminoacid sequence (“Fbh67067b”; SEQ ID NO:34) with the amino acid sequenceof mouse Potential Phospholipid-Transporting ATPase VA (mouseAT5A)(GenBank Accession No O54827; SEQ ID NO:47). The transmembrane domains(“TM1”, “TM2”, etc.), E1-E2 ATPases phosphorylation site(“phosphorylation site”), and phospholipid transporter specific aminoacid residues (“phospholipid transport”) are boxed.

FIG. 24 depicts a structural, hydrophobicity, and antigenicity analysisof the human 62092 polypeptide.

FIG. 25 depicts a multiple sequence alignment (MSA) of the amino acidsequences of the human 62092 protein (SEQ ID NO:40), human HINT (GenBankAccession No. NP_(—)005331; SEQ ID NO:48), and human FHIT (GenBankAccession No. NP_(—)002003; SEQ ID NO:49). The HIT family signaturemotifs are underlined and italicized. The location of the threehistidine residues of the histidine triad in human 62092 and human HINTare indicated by stars. The alignment was performed using the Clustalalgorithm which is part of the MegAlign™ program (e.g., version 3.1.7),which is part of the DNAStar™ sequence analysis software package. Thepairwise alignment parameters are as follows: K-tuple=1; Gap Penalty=3;Window=5; Diagonals saved=5. The multiple alignment parameters are asfollows: Gap Penalty=10; and Gap length penalty=10.

FIG. 26 depicts a structural, hydrophobicity, and antigenicity analysisof the HAAT polypeptide.

FIG. 27 depicts a Clustal W (1.74) alignment of the HAAT amino acidsequence (“Fbh58295FL”; SEQ ID NO:52) with the amino acid sequence ofrat amino acid system A transporter (ratATA2). The transmembrane domains(“TM1”, “TM2”, etc.) are boxed.

FIG. 28 depicts the results of a search which was performed against theMEMSAT database and which resulted in the identification of ten“transmembrane domains” in the HAAT amino acid sequence (SEQ ID NO:52).An additional predicted transmembrane domain (i.e., TM1) is also shown.

FIG. 29 depicts a structural, hydrophobicity, and antigenicity analysisof the human HST-4 polypeptide (SEQ ID NO:55).

FIG. 30 depicts a structural, hydrophobicity, and antigenicity analysisof the human HST-5 polypeptide (SEQ ID NO:58).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as “membrane transporter protein-1”or “MTP-1” nucleic acid and protein molecules, which are novel membersof a family of proteins possessing the ability to shuttle moleculesacross a lipid bilayer (e.g. to sequester, export or expel a pluralityof substances, for example, cytotoxic substances, metabolites, ions,and/or peptides, from the intracellular milieu). These novel moleculesare capable of transporting molecules (e.g., ions, proteins, and/orsmall molecules) across biological membranes and, thus, play a role inor function in a variety of cellular processes, e.g., maintenance ofcellular homeostasis.

As used herein, the term “transporter” includes a protein or molecule(e.g., a membrane-spanning protein or molecule) which is involved in themovement of a biochemical molecule from one side of a lipid bilayer tothe other, for example, against a preexisting concentration gradient.

Exemplary transporters, for example MTP-1 transporters, include at leastone, preferably two or three, more preferably four, five, six, seven,eight, nine, ten, eleven, more preferably about twelve “transmembranedomains” or more. As used herein, the term “transmembrane domain”includes an amino acid sequence of about 15 amino acid residues inlength which spans the plasma membrane. More preferably, a transmembranedomain includes about at least 20, 25, 30, 35, 40, or 45 amino acidresidues and spans the plasma membrane. Transmembrane domains are richin hydrophobic residues, and typically have an alpha-helical structure.In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or moreof the amino acids of a transmembrane domain are hydrophobic, e.g.,leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domainsare described in, for example, Zagotta W. N. et al., (1996) Annual Rev.Neurosci. 19: 235-263, the contents of which are incorporated herein byreference. Amino acid residues 23-40, 548-564, 588-612, 624-646,653-675, 1006-1023, 1236-1258, 1534-1556, 1587-1603, 1645-1667,1732-1749, 1931-1947 of the native MTP-1 protein are predicted tocomprise a transmembrane domain (see FIG. 1). Accordingly, MTP-1proteins having at least one transmembrane domain, preferably two orthree, more preferably four, five, six, seven, eight, nine, ten, elevenor twelve transmembrane domains selected from the group consisting ofamino acids 23-40, 548-564, 588-612, 624-646, 653-675, 1006-1023,1236-1258, 1534-1556, 1587-1603, 1645-1667, 1732-1749, 1931-1947 arewithin the scope of the invention. Also included within the scope of theinvention are MTP proteins having at least 50-60% homology, preferablyabout 60-70%, more preferably about 70-80%, or about 80-90% homologywith a transmembrane domain of human MTP-1 are within the scope of theinvention.

Preferably such MTP proteins comprise a family of MTP molecules. Theterm “family” when referring to the protein and nucleic acid moleculesof the invention is intended to mean two or more proteins or nucleicacid molecules having a common structural domain or motif and havingsufficient amino acid or nucleotide sequence homology as defined herein.Such family members can be naturally or non-naturally occurring and canbe from either the same or different species. For example, a family cancontain a first protein of human origin, as well as other, distinctproteins of human origin or alternatively, can contain homologues ofnon-human origin, e.g., monkey proteins. Members of a family may alsohave common functional characteristics.

In another embodiment, an MTP-1 molecule of the present invention isidentified based on the presence of at least one “ABC transporterdomain” in the protein or corresponding nucleic acid molecule. As usedherein, the term “ABC transporter domain” includes a protein domainhaving an amino acid sequence of about 131-232 amino acid residues and abit score of at least 80 when compared against an ABC transporter HiddenMarkov Model (HMM), e.g., PFAM accession number PF00005. In a preferredembodiment, an ABC transporter domain includes a protein domain havingan amino acid sequence of about 141-222 amino acid residues and a bitscore of at least 100. In another preferred embodiment, an ABCtransporter domain includes a protein domain having an amino acidsequence of about 151-212 amino acid residues and a bit score of atleast 120. Preferably, an ABC transporter domain includes a proteindomain having an amino acid sequence of about 171-192 amino acidresidues and a bit score of at least 140 (e.g., 144.2, 150, 160, 170,180, 190, 200, 206, 210 or more). To identify the presence of an ABCtransporter domain in an MTP-1 protein, the amino acid sequence of theprotein is used to search a database of known Hidden Markov Models (HMMse.g., the PFAM HMM database). The ABC transporter HMM has been assignedthe PFAM Accession PF00005 (http://pfam.wustl.edu), InterPro accessionnumber IPR0001617 (http://www.ebi.ac.uk/interpro), and Prosite accessionnumber PS00211 (http://www.expasy.ch/prosite). For example, a search wasperformed against the HMM database using the amino acid sequence (SEQ IDNO:2) of human MTP-1 resulting in the identification of a first ABCtransporter domain in the amino acid sequence of human MTP-1 (SEQ ID NO:2) at about residues 832-1012 having a score of 206.0, and a second ABCtransporter domain in the amino acid sequence of human MTP-1 (SEQ ID NO:2) at about residues 1818-1999 having a score of 144.2.

In a preferred embodiment, an ABC transporter domain as described hereinis characterized by the presence of an “ATP/AGP binding motif” and/or an“ABC transporter signature motif.” As used herein, the term “ATP/AGPbinding motif” includes a motif having the consensus sequence[AG]-X(4)-G-K-[ST] and is described under Prosite entry number PS00017(http://www.expasy.ch/prosite). ATP/AGP binding motifs can be found, forexample, within the first ABC transporter domains of the MTP-1 proteinof SEQ ID NO:2 at about residues 839-846 and within the second ABCtransporter domain of the MTP-1 protein of SEQ ID NO:2 at about residues1825-1832. As used herein, the term “ABC transporter signature motif”includes a protein motif having the consensus sequence[LIVMFYC]-[SA]-[SAPGLVFYKQH]-G-[DENQMW]-[KRQASPCLIMFW]-[KRNQSTAVM]-[KRACLVM]-[LIVMFYPAN]-{PHY}-[LIVMFW]-[SAGCLIVP]-{FYWHP}-{KRHP}-[LIVMFYWSTA]and is described under Prosite entry number PS00211(http://www.expasy.ch/prosite). An ABC transporter signature motif canbe found within the first ABC transporter domain of the MTP-1 protein orSEQ ID NO:2 at about residues 938-952. The consensus sequences describedherein are described according to standard Prosite Signature designation(e.g., all amino acids are indicated according to their universal singleletter designation; X designates any amino acid; X(n) designates any namino acids, e.g., X (2) designates any 2 amino acids; [LIVM] indicatesany one of the amino acids appearing within the brackets, e.g., any oneof L, I, V, or M, in the alternative, any one of Leu, Ile, Val, orMet.); and {LIVM} indicates any amino acid EXCEPT the amino acidsappearing within the brackets, e.g., not L, not I, not V, and not M.

Isolated proteins of the present invention, for example MTP-1 proteins,preferably have an amino acid sequence sufficiently identical to theamino acid sequence of SEQ ID NO:2, or are encoded by a nucleotidesequence sufficiently identical to SEQ ID NO: 1 or 3. As used herein,the term “sufficiently identical” refers to a first amino acid ornucleotide sequence which contains a sufficient or minimum number ofidentical or equivalent (e.g., an amino acid residue which has a similarside chain) amino acid residues or nucleotides to a second amino acid ornucleotide sequence such that the first and second amino acid ornucleotide sequences share common structural domains or motifs and/or acommon functional activity. For example, amino acid or nucleotidesequences which share common structural domains have at least 30%, 40%,or 50% homology, preferably 60% homology, more preferably 70%-80%, andeven more preferably 90-95% homology across the amino acid sequences ofthe domains and contain at least one and preferably two structuraldomains or motifs, are defined herein as sufficiently identical.Furthermore, amino acid or nucleotide sequences which share at least30%, 40%, or 50%, preferably 60%, more preferably 70-80%, or 90-95%homology and share a common functional activity are defined herein assufficiently identical.

As used interchangeably herein, an “MTP-1 activity”, “biologicalactivity of MTP-1” or “functional activity of MTP-1”, refers to anactivity exhibited by an MTP-1 protein, polypeptide or nucleic acidmolecule (e.g., in an MTP-1 expressing cell or tissue), on an MTP-1substrate, as determined in vivo, or in vitro, according to standardtechniques. In one embodiment, an MTP-1 activity is a direct activity,such as transport of an MTP-1-substrate. As used herein, a “MTP-1substrate” is a molecule which is transported from one side of abiological membrane to the other. Exemplary substrates include, but arenot limited to, cytotoxic substances, ions, peptides (e.g., antigenicpeptides, hormones, cytokines, neurotransmitters and the like), andmetabolites. Examples of MTP-1 substrates also include non-transportedmolecules that are essential for MTP-1 function, e.g., ATP or GTP.Alternatively, an MTP-1 activity is an indirect activity, such as acellular signaling activity mediated by the transport of an MTP-1substrate by MTP-1. In a preferred embodiment, the MTP-1 proteins of thepresent invention have one or more of the following activities: 1)modulate the import and/or export of MTP-1 substrates into or fromcells, e.g., peptides, ions, and/or metabolites, 2) modulate intra- orintercellular signaling, 3) removal of potentially harmful compounds(e.g., cytotoxic substances) from the cell, or facilitate thecompartmentalization of these molecules into a sequestered intracellularspace (e.g., the peroxisome), and 4) transport of biological moleculesacross membranes, e.g., the plasma membrane, or the membrane of themitochondrion, the peroxisome, the lysosome, the endoplasmic reticulum,the nucleus, or the vacuole.

Accordingly, another embodiment of the invention features isolated MTP-1proteins and polypeptides having an MTP-1 activity. Other preferredproteins are MTP-1 proteins having one or more of the following domains:a transmembrane domain, an ABC transporter domain and, preferably, anMTP-1 activity.

Additional preferred proteins have at least one transmembrane domain,one ABC transporter domain, and are, preferably, encoded by a nucleicacid molecule having a nucleotide sequence which hybridizes understringent hybridization conditions to a nucleic acid molecule comprisingthe nucleotide sequence of SEQ ID NO: 1 or 3.

The nucleotide sequence of the isolated human MTP-1 cDNA and thepredicted amino acid sequence of the human MTP-1 polypeptide are shownin SEQ ID NOs:1 and 2, respectively.

The human MTP-1 gene, which is approximately 6768 nucleotides in length,encodes a protein having a molecular weight of approximately 235.8 kDand which is approximately 2144 amino acid residues in length.

Various aspects of the invention are described in further detail inlater subsections.

Chapter II. 57312 and 53659, Novel Human Organic Anion TransporterMolecules and Uses Thereof

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel organic anion transporter family members, referred to herein as“Organic Anion Transporter” or “OAT” nucleic acid and protein molecules(e.g., OAT4 and OAT5). The OAT nucleic acid and protein molecules of thepresent invention are useful as modulating agents in regulating avariety of cellular processes, e.g., protection of cells and/or tissuesfrom organic anions, organic anion transport, inter- or intra-cellularsignaling, and/or hormonal responses. Accordingly, in one aspect, thisinvention provides isolated nucleic acid molecules encoding OAT proteinsor biologically active portions thereof, as well as nucleic acidfragments suitable as primers or hybridization probes for the detectionof OAT-encoding nucleic acids.

In one embodiment, the invention features an isolated nucleic acidmolecule that includes the nucleotide sequence set forth in SEQ ID NO:4,6, 7, or 9. In another embodiment, the invention features an isolatednucleic acid molecule that encodes a polypeptide including the aminoacid sequence set forth in SEQ ID NO:5 or 8.

In still other embodiments, the invention features isolated nucleic acidmolecules including nucleotide sequences that are substantiallyidentical (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.1% 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, or 99.9% identical) to the nucleotide sequence set forth as SEQID NO:4, 6, 7, or 9. The invention further features isolated nucleicacid molecules including at least 30 contiguous nucleotides of thenucleotide sequence set forth as SEQ ID NO:4, 6, 7, or 9. In anotherembodiment, the invention features isolated nucleic acid molecules whichencode a polypeptide including an amino acid sequence that issubstantially identical (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%,99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical) to the amino acidsequence set forth as SEQ ID NO:5 or 8. Also featured are nucleic acidmolecules which encode allelic variants of the polypeptide having theamino acid sequence set forth as SEQ ID NO:5 or 8. In addition toisolated nucleic acid molecules encoding full-length polypeptides, thepresent invention also features nucleic acid molecules which encodefragments, for example, biologically active or antigenic fragments, ofthe full-length polypeptides of the present invention (e.g., fragmentsincluding at least 10 contiguous amino acid residues of the amino acidsequence of SEQ ID NO:5 or 8). In still other embodiments, the inventionfeatures nucleic acid molecules that are complementary to, antisense to,or hybridize under stringent conditions to the isolated nucleic acidmolecules described herein.

In a related aspect, the invention provides vectors including theisolated nucleic acid molecules described herein (e.g., OAT-encodingnucleic acid molecules). Such vectors can optionally include nucleotidesequences encoding heterologous polypeptides. Also featured are hostcells including such vectors (e.g., host cells including vectorssuitable for producing OAT nucleic acid molecules and polypeptides).

In another aspect, the invention features isolated OAT polypeptidesand/or biologically active or antigenic fragments thereof. Exemplaryembodiments feature a polypeptide including the amino acid sequence setforth as SEQ ID NO:5 or 8, a polypeptide including an amino acidsequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, or 99.9% identical to the amino acid sequence set forth asSEQ ID NO:5 or 8, a polypeptide encoded by a nucleic acid moleculeincluding a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the nucleotidesequence set forth as SEQ ID NO:4, 6, 7, or 9. Also featured arefragments of the full-length polypeptides described herein (e.g.,fragments including at least 10, 15, 20, 25, 30, 35, 40, 45, or 50contiguous amino acid residues of the sequence set forth as SEQ ID NO:5or 8) as well as allelic variants of the polypeptide having the aminoacid sequence set forth as SEQ ID NO:5 or 8.

The OAT polypeptides and/or biologically active or antigenic fragmentsthereof, are useful, for example, as reagents or targets in assaysapplicable to treatment and/or diagnosis of OAT associated disorders. Inone embodiment, an OAT polypeptide or fragment thereof has an OATactivity. In another embodiment, an OAT polypeptide or fragment thereofhas at least one of the following domains: a transmembrane domain, asugar (and other) transporter domain, and/or an ATP/GTP-binding sitemotif A (P-loop) domain, and optionally, has an OAT activity. In arelated aspect, the invention features antibodies (e.g., antibodieswhich specifically bind to any one of the polypeptides, as describedherein) as well as fusion polypeptides including all or a fragment of apolypeptide described herein.

The present invention further features methods for detecting OATpolypeptides and/or OAT nucleic acid molecules, such methods featuring,for example, a probe, primer or antibody described herein. Also featuredare kits for the detection of OAT polypeptides and/or OAT nucleic acidmolecules. In a related aspect, the invention features methods foridentifying compounds which bind to and/or modulate the activity of anOAT polypeptide or OAT nucleic acid molecule described herein. Alsofeatured are methods for modulating an OAT activity.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel organic anion transporter family members, referred to herein as“Organic anion transporter” or “OAT” nucleic acid and protein molecules,e.g., OAT4 and OAT5. These novel molecules are capable of transportingorganic anions (e.g., drugs, xenobiotics, and/or metabolites oflipophilic compounds such as sulfate and glucuronide conjugates) acrosscellular membranes and, thus, play a role in or function in a variety ofcellular processes, e.g., protection of cells and/or tissues fromorganic anions, organic anion transport, inter- or intra-cellularsignaling, and/or hormonal responses. Thus, the OAT molecules of thepresent invention provide novel diagnostic targets and therapeuticagents to control organic anion transporter-associated disorders.

As used herein, an “organic anion transporter-associated disorder” or an“OAT-associated disorder” includes a disorder, disease or conditionwhich is caused or characterized by a misregulation (e.g.,downregulation or upregulation) of organic anion transporter activity.Organic anion transporter-associated disorders can detrimentally affectcellular functions such as cellular proliferation, growth,differentiation, or migration, inter- or intra-cellular communication;tissue function, such as cardiac function or musculoskeletal function;systemic responses in an organism, such as nervous system responses,hormonal responses (e.g., insulin response); immune responses; andprotection of cells from toxic compounds (e.g., carcinogens, toxins, ormutagens).

Examples of organic anion transporter-associated disorders include CNSdisorders such as cognitive and neurodegenerative disorders, examples ofwhich include, but are not limited to, Alzheimer's disease, dementiasrelated to Alzheimer's disease (such as Pick's disease), Parkinson's andother Lewy diffuse body diseases, senile dementia, Huntington's disease,Gilles de la Tourette's syndrome, multiple sclerosis, amyotrophiclateral sclerosis, progressive supranuclear palsy, epilepsy, andJakob-Creutzfieldt disease; autonomic function disorders such ashypertension and sleep disorders, and neuropsychiatric disorders, suchas depression, schizophrenia, schizoaffective disorder, korsakoff'spsychosis, mania, anxiety disorders, or phobic disorders; learning ormemory disorders, e.g., amnesia or age-related memory loss, attentiondeficit disorder, dysthymic disorder, major depressive disorder, mania,obsessive-compulsive disorder, psychoactive substance use disorders,anxiety, phobias, panic disorder, as well as bipolar affective disorder,e.g., severe bipolar affective (mood) disorder (BP-1), and bipolaraffective neurological disorders, e.g., migraine and obesity. FurtherCNS-related disorders include, for example, those listed in the AmericanPsychiatric Association's Diagnostic and Statistical manual of MentalDisorders (DSM), the most current version of which is incorporatedherein by reference in its entirety.

Further examples of organic anion transporter-associated disordersinclude cardiac-related disorders. Cardiovascular system disorders inwhich the OAT molecules of the invention may be directly or indirectlyinvolved include arteriosclerosis, ischemia reperfusion injury,restenosis, arterial inflammation, vascular wall remodeling, ventricularremodeling, rapid ventricular pacing, coronary microembolism,tachycardia, bradycardia, pressure overload, aortic bending, coronaryartery ligation, vascular heart disease, atrial fibrilation, Jervellsyndrome, Lange-Nielsen syndrome, long-QT syndrome, congestive heartfailure, sinus node dysfunction, angina, heart failure, hypertension,atrial fibrillation, atrial flutter, dilated cardiomyopathy, idiopathiccardiomyopathy, myocardial infarction, coronary artery disease, coronaryartery spasm, and arrhythmia. OAT-mediated or related disorders alsoinclude disorders of the musculoskeletal system such as paralysis andmuscle weakness, e.g., ataxia, myotonia, and myokymia.

Organic anion transporter disorders also include cellular proliferation,growth, differentiation, or migration disorders. Cellular proliferation,growth, differentiation, or migration disorders include those disordersthat affect cell proliferation, growth, differentiation, or migrationprocesses. As used herein, a “cellular proliferation, growth,differentiation, or migration process” is a process by which a cellincreases in number, size or content, by which a cell develops aspecialized set of characteristics which differ from that of othercells, or by which a cell moves closer to or further from a particularlocation or stimulus. The OAT molecules of the present invention areinvolved in signal transduction mechanisms, which are known to beinvolved in cellular growth, differentiation, and migration processes.Thus, the OAT molecules may modulate cellular growth, differentiation,or migration, and may play a role in disorders characterized byaberrantly regulated growth, differentiation, or migration. Suchdisorders include cancer, e.g., carcinoma, sarcoma, or leukemia; tumorangiogenesis and metastasis; skeletal dysplasia; hepatic disorders; andhematopoietic and/or myeloproliferative disorders.

OAT-associated or related disorders also include hormonal disorders,such as conditions or diseases in which the production and/or regulationof hormones in an organism is aberrant. Examples of such disorders anddiseases include type I and type II diabetes mellitus, pituitarydisorders (e.g., growth disorders), thyroid disorders (e.g.,hypothyroidism or hyperthyroidism), and reproductive or fertilitydisorders (e.g., disorders which affect the organs of the reproductivesystem, e.g., the prostate gland, the uterus, or the vagina; disorderswhich involve an imbalance in the levels of a reproductive hormone in asubject; disorders affecting the ability of a subject to reproduce; anddisorders affecting secondary sex characteristic development, e.g.,adrenal hyperplasia).

Further examples of OAT-associated or related disorders also includeimmune disorders, such as autoimmune disorders or immune deficiencydisorders, e.g., allergies, transplant rejection, responses topathogenic infection (e.g., bacterial, viral, or parasitic infection),lupus, multiple sclerosis, congenital X-linked infantilehypogammaglobulinemia, transient hypogammaglobulinemia, common variableimmunodeficiency, selective IgA deficiency, chronic mucocutaneouscandidiasis, or severe combined immunodeficiency.

DHDR-associated or related disorders also include viral disorders, i.e.,disorders affected or caused by infection by a virus, e.g., hepatitis,AIDS, certain cancers, influenza, and common colds.

OAT-associated or related disorders also include disorders affectingtissues in which OAT protein is expressed, e.g., the kidney,osteoblasts, brain cortex, lung, liver, bone marrow mononuclear cells(BM-MNC), and neutrophils.

The term “family” when referring to the protein and nucleic acidmolecules of the invention is intended to mean two or more proteins ornucleic acid molecules having a common structural domain or motif andhaving sufficient amino acid or nucleotide sequence homology as definedherein. Such family members can be naturally or non-naturally occurringand can be from either the same or different species. For example, afamily can contain a first protein of human origin as well as otherdistinct proteins of human origin or alternatively, can containhomologues of non-human origin, e.g., rat or mouse proteins. Members ofa family can also have common functional characteristics.

For example, the family of OAT proteins of the present inventioncomprises at least one “transmembrane domain”. As used herein, the term“transmembrane domain” includes an amino acid sequence of about 15 aminoacid residues in length which spans the plasma membrane. Morepreferably, a transmembrane domain includes about at least 20, 25, 30,35, 40, or 45 amino acid residues and spans the plasma membrane.Transmembrane domains are rich in hydrophobic residues, and typicallyhave an alpha-helical structure. In a preferred embodiment, at least50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of atransmembrane domain are hydrophobic, e.g., leucines, isoleucines,tyrosines, or tryptophans. Transmembrane domains are described in, forexample, Zagotta, W. N. et al. (1996) Annu. Rev. Neurosci. 19:235-263,the contents of which are incorporated herein by reference. Amino acidresidues 10-31,148-165, 172-195, 202-219, 228-252, 26-276, 347-365,375-399, 406-422, 431-451, 466-484, and 495-512 of the human OAT4protein are predicted to comprise transmembrane domains (see FIG. 4).Amino acid residues 106-130, 143-166, 174-191, 230-254, 265-284,314-335, 382-405, 419-443, 456-473, 579-603, 613-636, and 667-690 of thehuman OAT5 protein are predicted to comprise transmembrane domains (seeFIG. 3). Accordingly, OAT proteins having at least 50-60% homology,preferably about 60-70%, more preferably about 70-80%, or about 80-90%homology with a transmembrane domain of human OAT are within the scopeof the invention.

In another embodiment, members of the OAT family of proteins, include atleast one “sugar (and other) transporter domain” in the protein orcorresponding nucleic acid molecule. As used herein, the term “sugar(and other) transporter domain” includes a protein domain having atleast about 335-505 amino acid residues. Preferably, a sugar (and other)transporter domain includes a protein domain having an amino acidsequence of about 355-485, 375-465, 395-445, or more preferably about415-425 amino acid residues, and a bit score of at least 10, 20, 30, ormore preferably, 34.7. To identify the presence of a sugar (and other)transporter domain in an OAT protein, and make the determination that aprotein of interest has a particular profile, the amino acid sequence ofthe protein is searched against a database of known protein domains(e.g., the HMM database). The sugar (and other) transporter domain (HMM)has been assigned the PFAM Accession number PF00083 (see the PFAMwebsite, available online through Washington University in St. Louis). Asearch was performed against the HMM database resulting in theidentification of a sugar (and other) transporter domain in the aminoacid sequence of human OAT4 at about residues 103-527 of SEQ ID NO:5.Another search was performed against the HMM database, further resultingin the identification of a sugar (and other) transporter domain in theamino acid sequence of human OAT5 at about residues 141-555 of SEQ IDNO:8.

A description of the Pfam database can be found in Sonhammer et al.(1997) Proteins 28:405-420, and a detailed description of HMMs can befound, for example, in Gribskov et al. (1990) Meth. Enzymol.183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; andStultz et al. (1993) Protein Sci. 2:305-314, the contents of which areincorporated herein by reference.

In another embodiment, an OAT protein of the present invention includesat least one “ATP/GTP-binding site motif A (P-loop) domain”. As usedherein, the term “ATP/GTP-binding site motif A (P-loop) domain” includesan amino acid sequence having the consensus sequence [AG]-X(4)-G-K-[ST](SEQ ID NO: 11). ATP/GTP-binding site motif A (P-loop) domains aredescribed under Prosite entry PS00017 (see the Prosite website,available online through the Swiss Institute for Bioinformatics). Theconsensus sequence described herein is described according to thestandard Prosite signature designation (e.g., all amino acids areindicated according to their universal single letter designation; Xdesignates any amino acid; X(n) designates any n amino acids, e.g., X(4)designates any 4 amino acids; [AG] indicates any one of the amino acidsappearing within the brackets, e.g., any one of A or G). Searches wereperformed against the Prosite database resulting in the identificationof two ATP/GTP-binding site motif A (P-loop) domains in the amino acidsequence of OAT5 at about residues 343-350 and 360-367 of SEQ ID NO:8.

Isolated proteins of the present invention, preferably OAT proteins,have an amino acid sequence sufficiently homologous to the amino acidsequence of SEQ ID NO:5 or 8, or are encoded by a nucleotide sequencesufficiently homologous to SEQ ID NO:4, 6, 7, or 9. As used herein, theterm “sufficiently homologous” refers to a first amino acid ornucleotide sequence which contains a sufficient or minimum number ofidentical or equivalent (e.g., an amino acid residue which has a similarside chain) amino acid residues or nucleotides to a second amino acid ornucleotide sequence such that the first and second amino acid ornucleotide sequences share common structural domains or motifs and/or acommon functional activity. For example, amino acid or nucleotidesequences which share common structural domains having at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, 99.9% or more homology or identity across the amino acidsequences of the domains and contain at least one and preferably twostructural domains or motifs, are defined herein as sufficientlyhomologous. Furthermore, amino acid or nucleotide sequences which shareat least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,99.6%, 99.7%, 99.8%, 99.9% or more homology or identity and share acommon functional activity are defined herein as sufficientlyhomologous.

In a preferred embodiment, an OAT protein includes at least one of thefollowing domains: a transmembrane domain, a sugar (and other)transporter domain, and/or an ATP/GTP-binding site motif A (P-loop)domain, and has an amino acid sequence at least about 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or more homologous or identical to the amino acid sequence of SEQ IDNO:5 or 8. In yet another preferred embodiment, an OAT protein includesat least one of the following domains: a transmembrane domain, a sugar(and other) transporter domain, and/or an ATP/GTP-binding site motif A(P-loop) domain, and is encoded by a nucleic acid molecule having anucleotide sequence which hybridizes under stringent hybridizationconditions to a complement of a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:4, 6, 7, or 9. In another preferredembodiment, an OAT protein includes at least one of the followingdomains: a transmembrane domain, a sugar (and other) transporter domain,and/or an ATP/GTP-binding site motif A (P-loop) domain, and has an OATactivity.

As used interchangeably herein, an “OAT activity”, “biological activityof OAT” or “functional activity of OAT”, refers to an activity exhibitedby an OAT protein, polypeptide or nucleic acid molecule (e.g., in an OATexpressing cell or tissue) on an OAT responsive cell or an OATsubstrate, as determined in vivo or in vitro, according to standardtechniques. In one embodiment, an OAT activity is a direct activity,such as transport of an OAT substrate, e.g., a metabolite of alipophilic compound such as a sulfate or glucuronide conjugate. As usedherein, an “OAT substrate” is a molecule which is transported from oneside of a membrane to the other. Exemplary OAT substrates include, butare not limited to, organic anions such as drugs, xenobiotics, andmetabolites of lipophilic compounds such as sulfate and glucuronideconjugates. Examples of OAT substrates also include non-transportedmolecules that are essential for OAT function, such as ATP or GTP. AnOAT activity can also be a direct activity such as an association withan OAT target molecule. An OAT target molecule can be a non-OAT moleculeor an OAT protein or polypeptide of the present invention. In anexemplary embodiment, an OAT target molecule is an intracellularsignaling protein that mediates an OAT-modulated signal transductionpathway. An OAT activity can also be an indirect activity, such as acellular signaling activity mediated by transport of an OAT substrate orby interaction of the OAT protein with an OAT substrate or targetmolecule.

In a preferred embodiment, an OAT activity is at least one of thefollowing activities: (i) interaction with an OAT substrate or targetmolecule; (ii) transport of an OAT substrate across a membrane; (iii)interaction with and/or modulation of a second non-OAT protein; (iv)modulation of cellular signaling and/or gene transcription (e.g., eitherdirectly or indirectly); (v) protection of cells and/or tissues fromorganic anions; and/or (vi) modulation of hormonal responses.

The nucleotide sequence of the isolated human OAT4 cDNA and thepredicted amino acid sequence encoded by the OAT4 cDNA are shown in SEQID NO:4 and 5, respectively.

The nucleotide sequence of the isolated human OAT5 cDNA and thepredicted amino acid sequence encoded by the OAT5 cDNA are shown in SEQID NO:7 and 8, respectively.

The human OAT4 gene, which is approximately 2206 nucleotides in length,encodes a protein having a molecular weight of approximately 60.5 kD andwhich is approximately 550 amino acid residues in length. The human OAT5gene, which is approximately 2634 nucleotides in length, encodes aprotein having a molecular weight of approximately 79.6 kD and which isapproximately 724 amino acid residues in length.

Various aspects of the invention are described in further detail inlater subsections.

Chapter III. 57250, A Novel Human Sugar Transporter Family Member andUses Thereof

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel human sugar transporter family members, referred to herein as“human sugar transporter-1” or “HST-1” nucleic acid and polypeptidemolecules. The HST-1 nucleic acid and polypeptide molecules of thepresent invention are useful as modulating agents in regulating avariety of cellular processes, e.g., sugar homeostasis. Accordingly, inone aspect, this invention provides isolated nucleic acid moleculesencoding HST-1 polypeptides or biologically active portions thereof, aswell as nucleic acid fragments suitable as primers or hybridizationprobes for the detection of HST-1-encoding nucleic acids.

In one embodiment, the invention features an isolated nucleic acidmolecule that includes the nucleotide sequence set forth in SEQ ID NO:12 or 14. In another embodiment, the invention features an isolatednucleic acid molecule that encodes a polypeptide including the aminoacid sequence set forth in SEQ ID NO: 13.

In still other embodiments, the invention features isolated nucleic acidmolecules including nucleotide sequences that are substantiallyidentical (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more identical) to the nucleotide sequenceset forth as SEQ ID NO: 12 or 14. The invention further featuresisolated nucleic acid molecules including at least 50, 57, 63, 72, 100,124, 150, 172, 175, 200, 250, 268, 300, 305, 328, 350, 400, 431, 450,495, 500, 550, 600, 650, 700, 750, 800, 804, 850, 900, 950, 1000, 1050,1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750,1800, 1850, 1900 or more contiguous nucleotides of the nucleotidesequence set forth as SEQ ID NO: 12 or 14. In another embodiment, theinvention features isolated nucleic acid molecules which encode apolypeptide including an amino acid sequence that is substantiallyidentical (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% identical) to the amino acid sequence set forthas SEQ ID NO:13. The present invention also features nucleic acidmolecules which encode allelic variants of the polypeptide having theamino acid sequence set forth as SEQ ID NO:13. In addition to isolatednucleic acid molecules encoding full-length polypeptides, the presentinvention also features nucleic acid molecules which encode fragments,for example, biologically active or antigenic fragments, of thefull-length polypeptides of the present invention (e.g., fragmentsincluding at least 10, 20, 50, 100, 150, 155, 200, 250, 300, 350, 350,400, 450, 500 or more contiguous amino acid residues of the amino acidsequence of SEQ ID NO:13). In still other embodiments, the inventionfeatures nucleic acid molecules that are complementary to, antisense to,or hybridize under stringent conditions to the isolated nucleic acidmolecules described herein.

In another aspect, the invention provides vectors including the isolatednucleic acid molecules described herein (e.g., HST-1-encoding nucleicacid molecules). Such vectors can optionally include nucleotidesequences encoding heterologous polypeptides. Also featured are hostcells including such vectors (e.g., host cells including vectorssuitable for producing HST-1 nucleic acid molecules and polypeptides).

In another aspect, the invention features isolated HST-1 polypeptidesand/or biologically active or antigenic fragments thereof. Exemplaryembodiments feature a polypeptide including the amino acid sequence setforth as SEQ ID NO: 13, a polypeptide including an amino acid sequenceat least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the amino acid sequence setforth as SEQ ID NO: 13, a polypeptide encoded by a nucleic acid moleculeincluding a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical tothe nucleotide sequence set forth as SEQ ID NO: 12 or 14. Also featuredare fragments of the full-length polypeptides described herein (e.g.,fragments including at least 10, 20, 50, 100, 150, 155, 200, 250, 300,350, 350, 400, 450, 500 or more contiguous amino acid residues of thesequence set forth as SEQ ID NO: 13) as well as allelic variants of thepolypeptide having the amino acid sequence set forth as SEQ ID NO: 13.

The HST-1 polypeptides and/or biologically active or antigenic fragmentsthereof, are useful, for example, as reagents or targets in assaysapplicable to treatment and/or diagnosis of HST-1 mediated or relateddisorders. In one embodiment, an HST-1 polypeptide or fragment thereof,has an HST-1 activity. In another embodiment, an HST-1 polypeptide orfragment thereof, has a transmembrane domain and/or a sugar transporterfamily domain, and optionally, has an HST-1 activity. In a relatedaspect, the invention features antibodies (e.g., antibodies whichspecifically bind to any one of the polypeptides described herein) aswell as fusion polypeptides including all or a fragment of a polypeptidedescribed herein.

The present invention further features methods for detecting HST-1polypeptides and/or HST-1 nucleic acid molecules, such methodsfeaturing, for example, a probe, primer or antibody described herein.Also featured are kits e.g., kits for the detection of HST-1polypeptides and/or HST-1 nucleic acid molecules. In a related aspect,the invention features methods for identifying compounds which bind toand/or modulate the activity of an HST-1 polypeptide or HST-1 nucleicacid molecule described herein. Further featured are methods formodulating an HST-1 activity.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as “human sugar transporter-1” or“HST-1” nucleic acid and polypeptide molecules, which are novel membersof the sugar transporter family. These novel molecules are capable of,for example, modulating a transporter mediated activity (e.g., a sugartransporter mediated activity) in a cell, e.g., a liver cell, fat cell,muscle cell, or blood cell, such as an erythrocyte. These novelmolecules are capable of transporting molecules, e.g., monosaccharidessuch as D-glucose, D-fructose or D-galactose, across biologicalmembranes and, thus, play a role in or function in a variety of cellularprocesses, e.g., maintenance of sugar homeostasis.

As used herein, a “sugar transporter” includes a protein or polypeptidewhich is involved in transporting a molecule, e.g., a monosaccharidesuch as D-glucose, D-fructose or D-galactose, across the plasma membraneof a cell, e.g., a liver cell, fat cell, muscle cell, or blood cell,such as an erythrocyte. Sugar transporters regulate sugar homeostasis ina cell and, typically, have sugar substrate specificity. Examples ofsugar transporters include glucose transporters, fructose transporters,and galactose transporters.

As used herein, a “sugar transporter mediated activity” includes anactivity which involves a sugar transporter, e.g., a sugar transporterin a liver cell, fat cell, muscle cell, or blood cell, such as anerythrocyte. Sugar transporter mediated activities include the transportof sugars, e.g., D-glucose, D-fructose or D-galactose, into and out ofcells; the stimulation of molecules that regulate glucose homeostasis(e.g., insulin and glucagon), in cells, e.g., pancreatic cells; and theparticipation in signal transduction pathways associated with sugarmetabolism.

As the HST-1 molecules of the present invention are sugar transporters,they may be useful for developing novel diagnostic and therapeuticagents for sugar transporter associated disorders. As used herein, theterm “sugar transporter associated disorder” includes a disorder,disease, or condition which is characterized by an aberrant, e.g.,upregulated or downregulated, sugar transporter mediated activity. Sugartransporter associated disorders typically result in, for example,upregulated or downregulated, sugar levels in a cell. Examples of sugartransporter associated disorders include disorders associated with sugarhomeostasis, such as obesity, anorexia, type-1 diabetes, type-2diabetes, hypoglycemia, glycogen storage disease (Von Gierke disease),type I glycogenosis, bipolar disorder, seasonal affective disorder, andcluster B personality disorders. HST-1-associated disorders may alsoinclude cellular growth or proliferation disorders. Further examples ofsugar transporter associated disorders include cellular growth orproliferation disorders, such as cancer, e.g., carcinoma, sarcoma, orleukemia, examples of which include, but are not limited to, colon,ovarian, lung, breast, endometrial, uterine, hepatic, gastrointestinal,prostate, and brain cancer; tumorigenesis and metastasis; skeletaldysplasia; and hematopoietic and/or myeloproliferative disorders.

The term “family” when referring to the polypeptide and nucleic acidmolecules of the invention is intended to mean two or more polypeptidesor nucleic acid molecules having a common structural domain or motif andhaving sufficient amino acid or nucleotide sequence homology as definedherein. Such family members can be naturally or non-naturally occurringand can be from either the same or different species. For example, afamily can contain a first polypeptide of human origin, as well asother, distinct polypeptides of human origin or alternatively, cancontain homologues of non-human origin, e.g., mouse or monkeypolypeptides. Members of a family may also have common functionalcharacteristics.

For example, the family of HST-1 polypeptides comprise at least one“transmembrane domain” and preferably twelve transmembrane domains. Asused herein, the term “transmembrane domain” includes an amino acidsequence of about 20-45 amino acid residues in length which spans theplasma membrane. More preferably, a transmembrane domain includes aboutat least 20, 25, 30, or 35 amino acid residues and spans the plasmamembrane. Transmembrane domains are rich in hydrophobic residues, andtypically have an alpha-helical structure. In a preferred embodiment, atleast 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of atransmembrane domain are hydrophobic, e.g., leucines, isoleucines,alanines, valines, phenylalanines, prolines or methionines.Transmembrane domains are described in, for example, Zagotta W. N. etal, (1996) Annual Rev. Neurosci. 19: 235-263, the contents of which areincorporated herein by reference. A MEMSAT analysis resulted in theidentification of twelve transmembrane domains in the amino acidsequence of human HST-1 (SEQ ID NO:13) at about residues 20-36, 150-167,174-196, 204-220, 231-255, 263-282, 355-372, 387-405, 413-431, 438-462,469-485, and 505-521 as set forth in FIG. 8.

Accordingly, HST-1 polypeptides having at least 50-60% homology,preferably about 60-70%, more preferably about 70-80%, or about 80-90%homology with a transmembrane domain of human HST-1 are within the scopeof the invention.

In another embodiment, an HST-1 molecule of the present invention isidentified based on the presence of at least one “sugar transporterfamily domain.” As used herein, the term “sugar transporter familydomain” includes a protein domain having at least about 350-500 aminoacid residues and a sugar transporter mediated activity. Preferably, asugar transporter family domain includes a polypeptide having an aminoacid sequence of about 350-450, 400-450, or more preferably, about 419amino acid residues and a sugar transporter mediated activity. Toidentify the presence of a sugar transporter family domain in an HST-1protein, and make the determination that a protein of interest has aparticular profile, the amino acid sequence of the protein may besearched against a database of known protein domains (e.g., the PFAM HMMdatabase). A PFAM sugar transporter family domain has been assigned thePFAM Accession PF00083. A search was performed against the PFAM HMMdatabase resulting in the identification of a sugar transporter familydomain in the amino acid sequence of human HST-1 (SEQ ID NO: 13) atabout residues 117-536 of SEQ ID NO: 13.

Preferably a “sugar transporter family domain” has a “sugar transportermediated activity” as described herein. For example, a sugar transporterfamily domain may have the ability to bind a monosaccharide, such asD-glucose, D-fructose, and/or D-galactose; the ability to transport amonosaccharide such as D-glucose, D-fructose, and/or D-galactose, acrossa cell membrane (e.g., a liver cell membrane, fat cell membrane, musclecell membrane, and/or blood cell membrane, such as an erythrocytemembrane); and the ability to modulate sugar homeostasis in a cell.Accordingly, identifying the presence of a “sugar transporter familydomain” can include isolating a fragment of an HST-1 molecule (e.g., anHST-1 polypeptide) and assaying for the ability of the fragment toexhibit one of the aforementioned sugar transporter mediated activities.

A description of the Pfam database can be found in Sonhammer et al.(1997) Proteins 28:405-420 and a detailed description of HMMs can befound, for example, in Gribskov et al. (1990) Meth. Enzymol.183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA84:4355-4358;. Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; andStultz et al. (1993) Protein Sci. 2:305-314, the contents of which areincorporated herein by reference.

In a preferred embodiment, the NPM-1 molecules of the invention includeat least one, preferably two, even more preferably twelve transmembranedomain(s) and/or at least one sugar transporter family domain.

Isolated polypeptides of the present invention, preferably HST-1polypeptides, have an amino acid sequence sufficiently identical to theamino acid sequence of SEQ ID NO: 13 or are encoded by a nucleotidesequence sufficiently identical to SEQ ID NO: 12 or 14. As used herein,the term “sufficiently identical” refers to a first amino acid ornucleotide sequence which contains a sufficient or minimum number ofidentical or equivalent (e.g., an amino acid residue which has a similarside chain) amino acid residues or nucleotides to a second amino acid ornucleotide sequence such that the first and second amino acid ornucleotide sequences share common structural domains or motifs and/or acommon functional activity. For example, amino acid or nucleotidesequences which share common structural domains having at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more homology or identity across the amino acidsequences of the domains and contain at least one and preferably twostructural domains or motifs, are defined herein as sufficientlyidentical. Furthermore, amino acid or nucleotide sequences which shareat least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology or identity and sharea common functional activity are defined herein as sufficientlyidentical.

In a preferred embodiment, an HST-1 polypeptide includes at least one ormore of the following domains: a transmembrane domain and/or a sugartransporter family domain, and has an amino acid sequence at least about50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more homologous or identical to the aminoacid sequence of SEQ ID NO: 13. In yet another preferred embodiment, anHST-1 polypeptide includes at least one or more of the followingdomains: a transmembrane domain and/or a sugar transporter familydomain, and is encoded by a nucleic acid molecule having a nucleotidesequence which hybridizes under stringent hybridization conditions to acomplement of a nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO: 12 or 14. In another preferred embodiment, an HST-1polypeptide includes at least one or more of the following domains: atransmembrane domain and/or a sugar transporter family domain, and hasan HST-1 activity.

As used interchangeably herein, an “HST-1 activity”, “biologicalactivity of HST-1” or “functional activity of HST-1,” refers to anactivity exerted by an HST-1 polypeptide or nucleic acid molecule on anHST-1 responsive cell or tissue, or on an HST-1 polypeptide substrate,as determined in vivo, or in vitro, according to standard techniques. Inone embodiment, an HST-1 activity is a direct activity, such as anassociation with an HST-1-target molecule. As used herein, a“substrate,” “target molecule,” or “binding partner” is a molecule withwhich an HST-1 polypeptide binds or interacts in nature, such thatHST-1-mediated function is achieved. An HST-1 target molecule can be anon-HST-1 molecule or an HST-1 polypeptide or polypeptide of the presentinvention. In an exemplary embodiment, an HST-1 target molecule is anHST-1 ligand, e.g., a sugar transporter ligand such as D-glucose,D-fructose, and/or D-galactose. Alternatively, an HST-1 activity is anindirect activity, such as a cellular signaling activity mediated byinteraction of the HST-1 polypeptide with an HST-1 ligand. Thebiological activities of HST-1 are described herein. For example, theHST-1 polypeptides of the present invention can have one or more of thefollowing activities: (1) maintain sugar homeostasis in a cell, (2)influence insulin and/or glucagon secretion, (3) bind a monosaccharide,e.g., D-glucose, D-fructose, and/or D-galactose, and/or (4) transportmonosaccharides across a cell membrane.

The nucleotide sequence of the isolated human HST-1 cDNA and thepredicted amino acid sequence of the human HST-1 polypeptide are shownin SEQ ID NOs:12 and 14, respectively.

The human HST-1 gene, which is approximately 1917 nucleotides in length,encodes a polypeptide which is approximately 572 amino acid residues inlength.

Various aspects of the invention are described in further detail inlater subsections.

Chapter IV. 63760, A Novel Human Transporter and Uses Thereof

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel human transporter family members, referred to herein as“transporter-2” or “TP-2” nucleic acid and polypeptide molecules. TheTP-2 nucleic acid and polypeptide molecules of the present invention areuseful as modulating agents in regulating a variety of cellularprocesses, e.g., cellular growth, migration, or proliferation.Accordingly, in one aspect, this invention provides isolated nucleicacid molecules encoding TP-2 polypeptides or biologically activeportions thereof, as well as nucleic acid fragments suitable as primersor hybridization probes for the detection of TP-2-encoding nucleicacids.

In one embodiment, the invention features an isolated nucleic acidmolecule that includes the nucleotide sequence set forth in SEQ ID NO:15or 17. In another embodiment, the invention features an isolated nucleicacid molecule that encodes a polypeptide including the amino acidsequence set forth in SEQ ID NO: 16.

In still other embodiments, the invention features isolated nucleic acidmolecules including nucleotide sequences that are substantiallyidentical (e.g., 60% identical) to the nucleotide sequence set forth asSEQ ID NO:15 or 17. The invention further features isolated nucleic acidmolecules including at least 50 contiguous nucleotides of the nucleotidesequence set forth as SEQ ID NO:15 or 17. In another embodiment, theinvention features isolated nucleic acid molecules which encode apolypeptide including an amino acid sequence that is substantiallyidentical (e.g., 60% identical) to the amino acid sequence set forth asSEQ ID NO: 16. The present invention also features nucleic acidmolecules which encode allelic variants of the polypeptide having theamino acid sequence set forth as SEQ ID NO: 16. In addition to isolatednucleic acid molecules encoding full-length polypeptides, the presentinvention also features nucleic acid molecules which encode fragments,for example, biologically active or antigenic fragments, of thefull-length polypeptides of the present invention (e.g., fragmentsincluding at least 10 contiguous amino acid residues of the amino acidsequence of SEQ ID NO: 16). In still other embodiments, the inventionfeatures nucleic acid molecules that are complementary to, antisense to,or hybridize under stringent conditions to the isolated nucleic acidmolecules described herein.

In another aspect, the invention provides vectors including the isolatednucleic acid molecules described herein (e.g., TP-2-encoding nucleicacid molecules). Such vectors can optionally include nucleotidesequences encoding heterologous polypeptides. Also featured are hostcells including such vectors (e.g., host cells including vectorssuitable for producing TP-2 nucleic acid molecules and polypeptides).

In another aspect, the invention features isolated TP-2 polypeptidesand/or biologically active or antigenic fragments thereof. Exemplaryembodiments feature a polypeptide including the amino acid sequence setforth as SEQ ID NO: 16, a polypeptide including an amino acid sequenceat least 60% identical to the amino acid sequence set forth as SEQ IDNO:16, a polypeptide encoded by a nucleic acid molecule including anucleotide sequence at least 60% identical to the nucleotide sequenceset forth as SEQ ID NO: 15 or 17. Also featured are fragments of thefull-length polypeptides described herein (e.g., fragments including atleast 10 contiguous amino acid residues of the sequence set forth as SEQID NO: 16) as well as allelic variants of the polypeptide having theamino acid sequence set forth as SEQ ID NO:16.

The TP-2 polypeptides and/or biologically active or antigenic fragmentsthereof, are useful, for example, as reagents or targets in assaysapplicable to treatment and/or diagnosis of TP-2 mediated or relateddisorders. In one embodiment, a TP-2 polypeptide or fragment thereof,has a TP-2 activity. In another embodiment, a TP-2 polypeptide orfragment thereof, includes at least one of the following domains: atransmembrane domain, a sugar transporter domain, a LacY proton/sugarsymporter domain, and optionally, has a TP-2 activity. In a relatedaspect, the invention features antibodies (e.g., antibodies whichspecifically bind to any one of the polypeptides described herein) aswell as fusion polypeptides including all or a fragment of a polypeptidedescribed herein.

The present invention further features methods for detecting TP-2polypeptides and/or TP-2 nucleic acid molecules, such methods featuring,for example, a probe, primer or antibody described herein. Also featuredare kits e.g., kits for the detection of TP-2 polypeptides and/or TP-2nucleic acid molecules. In a related aspect, the invention featuresmethods for identifying compounds which bind to and/or modulate theactivity of a TP-2 polypeptide or TP-2 nucleic acid molecule describedherein. Further featured are methods for modulating a TP-2 activity.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as “transporter-2” or “TP-2” nucleicacid and polypeptide molecules, which are novel members of thetransporter family. These novel molecules are capable of, for example,transporting ions, proteins, sugars, and small molecules acrossbiological membranes both within a cell and between the cell and theenvironment and, thus, play a role in or function in a variety ofcellular processes, e.g., proliferation, growth, differentiation,migration, immune responses, hormonal responses, and inter- orintra-cellular communication.

As used herein, the term “transporter” includes a molecule which isinvolved in the movement of a biochemical molecule from one side of alipid bilayer to the other, for example, against a pre-existingconcentration gradient. Transporters are usually involved in themovement of biochemical compounds which would normally not be able tocross a membrane (e.g., a protein; an ion; a sugar; or other smallmolecule, such as ATP; signaling molecules; vitamins; and cofactors).Transporter molecules are involved in the growth, development, anddifferentiation of cells, in the regulation of cellular homeostasis, inthe metabolism and catabolism of biochemical molecules necessary forenergy production or storage, in intra- or inter-cellular signaling, inmetabolism or catabolism of metabolically important biomolecules, and inthe removal of potentially harmful compounds from the interior of thecell. Examples of transporters include GSH transporters, ATPtransporters, sugar transporters, and fatty acid transporters. Astransporters, the TP-2 molecules of the present invention provide noveldiagnostic targets and therapeutic agents to controltransporter-associated disorders.

As used herein, a “transporter-associated disorder” includes a disorder,disease or condition which is caused or characterized by a misregulation(e.g., downregulation or upregulation) of a transporter-mediatedactivity. Transporter-associated disorders can detrimentally affectcellular functions such as cellular proliferation, growth,differentiation, or migration, cellular regulation of homeostasis,inter- or intra-cellular communication; tissue function, such as cardiacfunction or musculoskeletal function; systemic responses in an organism,such as nervous system responses, hormonal responses (e.g., insulinresponse), or immune responses; and protection of cells from toxiccompounds (e.g., carcinogens, toxins, mutagens, and toxic byproducts ofmetabolic activity (e.g., reactive oxygen species)). Examples oftransporter-associated disorders include CNS disorders such as cognitiveand neurodegenerative disorders, examples of which include, but are notlimited to, Alzheimer's disease, dementias related to Alzheimer'sdisease (such as Pick's disease), Parkinson's and other Lewy diffusebody diseases, senile dementia, Huntington's disease, Gilles de laTourette's syndrome, multiple sclerosis, amyotrophic lateral sclerosis,progressive supranuclear palsy, epilepsy, and Jakob-Creutzfieldtdisease; autonomic function disorders such as hypertension and sleepdisorders, and neuropsychiatric disorders, such as depression,schizophrenia, schizoaffective disorder, korsakoff's psychosis, mania,anxiety disorders, or phobic disorders; learning or memory disorders,e.g., amnesia or age-related memory loss, attention deficit disorder,dysthymic disorder, major depressive disorder, mania,obsessive-compulsive disorder, psychoactive substance use disorders,anxiety, phobias, panic disorder, as well as bipolar affective disorder,e.g., severe bipolar affective (mood) disorder (BP-1), and bipolaraffective neurological disorders, e.g., migraine and obesity. FurtherCNS-related disorders include, for example, those listed in the AmericanPsychiatric Association's Diagnostic and Statistical manual of MentalDisorders (DSM), the most current version of which is incorporatedherein by reference in its entirety.

Further examples of transporter-associated disorders includecardiac-related disorders. Cardiovascular system disorders in which theTP-2 molecules of the invention may be directly or indirectly involvedinclude arteriosclerosis, ischemia reperfusion injury, restenosis,arterial inflammation, vascular wall remodeling, ventricular remodeling,rapid ventricular pacing, coronary microembolism, tachycardia,bradycardia, pressure overload, aortic bending, coronary arteryligation, vascular heart disease, atrial fibrilation, Jervell syndrome,Lange-Nielsen syndrome, long-QT syndrome, congestive heart failure,sinus node dysfunction, angina, heart failure, hypertension, atrialfibrillation, atrial flutter, dilated cardiomyopathy, idiopathiccardiomyopathy, myocardial infarction, coronary artery disease, coronaryartery spasm, and arrhythmia. TP-2-mediated or related disorders alsoinclude disorders of the musculoskeletal system such as paralysis andmuscle weakness, e.g., ataxia, myotonia, and myokymia.

Transporter-associated disorders also include cellular proliferation,growth, differentiation, or migration disorders. Cellular proliferation,growth, differentiation, or migration disorders include those disordersthat affect cell proliferation, growth, differentiation, or migrationprocesses. As used herein, a “cellular proliferation, growth,differentiation, or migration process” is a process by which a cellincreases in number, size or content, by which a cell develops aspecialized set of characteristics which differ from that of othercells, or by which a cell moves closer to or further from a particularlocation or stimulus. The TP-2 molecules of the present invention areinvolved in signal transduction mechanisms, which are known to beinvolved in cellular growth, differentiation, and migration processes.Thus, the TP-2 molecules may modulate cellular growth, differentiation,or migration, and may play a role in disorders characterized byaberrantly regulated growth, differentiation, or migration. Suchdisorders include cancer, e.g., carcinoma, sarcoma, or leukemia; tumorangiogenesis and metastasis; skeletal dysplasia; hepatic disorders; andhematopoietic and/or myeloproliferative disorders.

TP-2-associated disorders also include hormonal disorders, such asconditions or diseases in which the production and/or regulation ofhormones in an organism is aberrant. Examples of such disorders anddiseases include type I and type II diabetes mellitus, pituitarydisorders (e.g., growth disorders), thyroid disorders (e.g.,hypothyroidism or hyperthyroidism), and reproductive or fertilitydisorders (e.g., disorders which affect the organs of the reproductivesystem, e.g., the prostate gland, the uterus, or the vagina; disorderswhich involve an imbalance in the levels of a reproductive hormone in asubject; disorders affecting the ability of a subject to reproduce; anddisorders affecting secondary sex characteristic development, e.g.,adrenal hyperplasia).

TP-2-associated disorders also include immune disorders, such asautoimmune disorders or immune deficiency disorders, e.g., congenitalX-linked infantile hypogammaglobulinemia, transienthypogammaglobulinemia, common variable immunodeficiency, selective IgAdeficiency, chronic mucocutaneous candidiasis, or severe combinedimmunodeficiency.

TP-2-associated disorders also include disorders associated with sugarhomeostasis, such as obesity, anorexia, hypoglycemia, glycogen storagedisease (Von Gierke disease), type I glycogenosis, seasonal affectivedisorder, and cluster B personality disorders.

TP-2-associated disorders also include disorders affecting tissues inwhich TP-2 protein is expressed.

As used herein, a “transporter-mediated activity” includes an activitywhich involves the facilitated movement of one or more molecules fromone side of a biological membrane to the other. Transporter-mediatedactivities include the import or export across internal or externalcellular membranes of biochemical molecules necessary for energyproduction or storage, intra- or inter-cellular signaling, metabolism orcatabolism of metabolically important biomolecules, and removal ofpotentially harmful compounds from the cell.

The term “family” when referring to the polypeptide and nucleic acidmolecules of the invention is intended to mean two or more polypeptidesor nucleic acid molecules having a common structural domain or motif andhaving sufficient amino acid or nucleotide sequence homology as definedherein. Such family members can be naturally or non-naturally occurringand can be from either the same or different species. For example, afamily can contain a first polypeptide of human origin, as well asother, distinct polypeptides of human origin or alternatively, cancontain homologues of non-human origin, e.g., mouse or monkeypolypeptides. Members of a family may also have common functionalcharacteristics.

For example, the family of TP-2 polypeptides comprise at least one“transmembrane domain” and preferably twelve transmembrane domains. Asused herein, the term “transmembrane domain” includes an amino acidsequence of about 15-45 amino acid residues in length which spans theplasma membrane. More preferably, a transmembrane domain includes aboutat least 15, 20, 25, 30, 35, 40, or 45 amino acid residues and spans theplasma membrane. Transmembrane domains are rich in hydrophobic residues,and typically have an alpha-helical structure. In a preferredembodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the aminoacids of a transmembrane domain are hydrophobic, e.g., leucines,isoleucines, alanines, valines, phenylalanines, prolines or methionines.Transmembrane domains are described in, for example, Zagotta W. N. etal, (1996) Annual Rev. Neurosci. 19: 235-263, the contents of which areincorporated herein by reference. A MEMSAT analysis and a structural,hydrophobicity, and antigenicity analysis resulted in the identificationof twelve transmembrane domains in the amino acid sequence of human TP-2(SEQ ID NO: 16) at about residues 45-69, 80-102, 112-126, 133-156,167-190, 197-218, 288-310, 323-343, 352-368, 375-391, 409-433, and442-458 as set forth in FIGS. 11 and 12.

Accordingly, TP-2 polypeptides having at least 50-60% homology,preferably about 60-70%, more preferably about 70-80%, or about 80-90%homology with a transmembrane domain of human TP-2 are within the scopeof the invention.

For example, in one embodiment, members of the TP-2 family of proteinsinclude at least one “sugar transporter domain” in the protein orcorresponding nucleic acid molecule. As used herein, the term “sugartransporter domain” includes a protein domain having at least about350-500 amino acid residues and a sugar transporter mediated activity.Preferably, a sugar transporter domain includes a polypeptide having anamino acid sequence of about 350-450, 400-450, or more preferably about417 amino acid residues, and a sugar transporter mediated activity. Toidentify the presence of a sugar transporter domain in a TP-2 protein,and make the determination that a protein of interest has a particularprofile, the amino acid sequence of the protein may be searched againsta database of known protein domains (e.g., the PFAM HMM database). APFAM sugar transporter domain has been assigned the PFAM AccessionPF00083. A search was performed against the PFAM HMM database resultingin the identification of a sugar transporter domain in the amino acidsequence of human TP-2 (SEQ ID NO:16) at about residues 37-454 of SEQ IDNO:16.

As used herein, a “sugar transporter mediated activity” includes theability to bind a monosaccharide, such as D-glucose, D-fructose, and/orD-galactose; the ability to transport a monosaccharide such asD-glucose, D-fructose, and/or D-galactose, across a cell membrane (e.g.,a liver cell membrane, fat cell membrane, muscle cell membrane, and/orblood cell membrane, such as an erythrocyte membrane); and the abilityto modulate sugar homeostasis in a cell. Accordingly, identifying thepresence of a “sugar transporter domain” can include isolating afragment of a TP-2 molecule (e.g., a TP-2 polypeptide) and assaying forthe ability of the fragment to exhibit one of the aforementioned sugartransporter mediated activities.

In another embodiment, a TP-2 molecule of the present invention isidentified based on the presence of at least one “LacY proton/sugarsymporter domain.” As used herein, the term “LacY proton/sugar symporterdomain” includes a protein domain having at least about 350-500 aminoacid residues and a LacY proton/sugar symporter mediated activity.Preferably, a LacY proton/sugar symporter domain includes a proteindomain having an amino acid sequence of about 300-400, 300-350, or morepreferably, about 344 amino acid residues and a LacY proton/sugarsymporter mediated activity. To identify the presence of a LacYproton/sugar symporter domain in a TP-2 protein, and make thedetermination that a protein of interest has a particular profile, theamino acid sequence of the protein may be searched against a database ofknown protein domains (e.g., the PFAM HMM database). A PFAM LacYproton/sugar symporter domain has been assigned the PFAM AccessionPF01306. A search was performed against the PFAM HMM database resultingin the identification of a LacY proton/sugar symporter domain in theamino acid sequence of human TP-2 (SEQ ID NO:16) at about residues39-383 of SEQ ID NO:16.

As used herein, a “LacY proton/sugar symporter mediated activity”includes the ability to mediate the transport of a variety of sugars(e.g., D-glucose, D-fructose, and/or D-galactose) with the concomitanttransport of hydrogen ions across a biological membrane. Accordingly,identifying the presence of a “LacY proton/sugar symporter domain” caninclude isolating a fragment of a TP-2 molecule (e.g., a TP-2polypeptide) and assaying for the ability of the fragment to exhibit oneof the aforementioned LacY proton/sugar symporter mediated activities.

A description of the Pfam database can be found in Sonhammer et al.(1997) Proteins 28:405-420 and a detailed description of HMMs can befound, for example, in Gribskov et al. (1990) Meth. Enzymol.183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; andStultz et al. (1993) Protein Sci. 2:305-314, the contents of which areincorporated herein by reference.

In a preferred embodiment, the TP-2 molecules of the invention includeat least one, preferably two, even more preferably twelve transmembranedomain(s), and/or at least one sugar transporter domain, and/or at leastone LacY proton/sugar symporter domain.

Isolated polypeptides of the present invention, preferably TP-2polypeptides, have an amino acid sequence sufficiently identical to theamino acid sequence of SEQ ID NO:16 or are encoded by a nucleotidesequence sufficiently identical to SEQ ID NO: 15 or 17. As used herein,the term “sufficiently identical” refers to a first amino acid ornucleotide sequence which contains a sufficient or minimum number ofidentical or equivalent (e.g., an amino acid residue which has a similarside chain) amino acid residues or nucleotides to a second amino acid ornucleotide sequence such that the first and second amino acid ornucleotide sequences share common structural domains or motifs and/or acommon functional activity. For example, amino acid or nucleotidesequences which share common structural domains having at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, ormore homology or identity across the amino acid sequences of the domainsand contain at least one and preferably two structural domains ormotifs, are defined herein as sufficiently identical. Furthermore, aminoacid or nucleotide sequences which share at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologyor identity and share a common functional activity are defined herein assufficiently identical.

In a preferred embodiment, a TP-2 polypeptide includes at least one ormore of the following domains: a transmembrane domain, and/or a sugartransporter domain, and/or a LacY proton/sugar symporter domain, and hasan amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more homologous or identicalto the amino acid sequence of SEQ ID NO: 16. In yet another preferredembodiment, a TP-2 polypeptide includes at least one or more of thefollowing domains: a transmembrane domain, and/or a sugar transporterdomain, and/or a LacY proton/sugar symporter domain, and is encoded by anucleic acid molecule having a nucleotide sequence which hybridizesunder stringent hybridization conditions to a complement of a nucleicacid molecule comprising the nucleotide sequence of SEQ ID NO:15 or 17.In another preferred embodiment, a TP-2 polypeptide includes at leastone or more of the following domains: a transmembrane domain, and/or asugar transporter domain, and/or a LacY proton/sugar symporter domain,and has a TP-2 activity.

As used interchangeably herein, a “TP-2 activity”, “biological activityof TP-2” or “functional activity of TP-2”, refers to an activity exertedby a TP-2 protein, polypeptide or nucleic acid molecule on a TP-2responsive cell or tissue, or on a TP-2 protein substrate, as determinedin vivo, or in vitro, according to standard techniques. In oneembodiment, a TP-2 activity is a direct activity, such as an associationwith a TP-2-target molecule. As used herein, a “target molecule” or“binding partner” is a molecule with which a TP-2 protein binds orinteracts in nature, such that TP-2-mediated function is achieved. ATP-2 target molecule can be a non-TP-2 molecule or a TP-2 protein orpolypeptide of the present invention (e.g., a molecule to betransported, e.g., a monosaccharide). In an exemplary embodiment, a TP-2target molecule is a TP-2 ligand (e.g., an energy molecule, ametabolite, a monosaccharide or an ion). Alternatively, a TP-2 activityis an indirect activity, such as a cellular signaling activity mediatedby interaction of the TP-2 protein with a TP-2 ligand. The biologicalactivities of TP-2 are described herein. For example, the TP-2 proteinsof the present invention can have one or more of the followingactivities: 1) modulate the import and export of molecules, e.g.,hormones, ions, cytokines, neurotransmitters, monosaccharides, andmetabolites, from cells, 2) modulate intra- or inter-cellular signaling,3) modulate removal of potentially harmful compounds from the cell, orfacilitate the compartmentalization of these molecules into asequestered intra-cellular space (e.g., the peroxisome), and 4) modulatetransport of biological molecules across membranes, e.g., the plasmamembrane, or the membrane of the mitochondrion, the peroxisome, thelysosome, the endoplasmic reticulum, the nucleus, or the vacuole.

The nucleotide sequence of the isolated human TP-2 cDNA and thepredicted amino acid sequence of the human TP-2 polypeptide are shown inSEQ ID NOs:15 and 16, respectively.

The human TP-2 gene, which is approximately 1963 nucleotides in length,encodes a polypeptide which is approximately 474 amino acid residues inlength.

Various aspects of the invention are described in further detail inlater subsections.

Chapter V. 49938, A Novel Human Phospholipid Transporter and UsesTherefor

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel phospholipid transporter family members, referred to herein as“Phospholipid Transporter-1” or “PLTR-1” nucleic acid and proteinmolecules. The PLTR-1 nucleic acid and protein molecules of the presentinvention are useful as modulating agents in regulating a variety ofcellular processes, e.g., phospholipid transport (e.g.,aminophospholipid transport), absorption, secretion, gene expression,intra- or intercellular signaling, blood coagulation, and/or cellularproliferation, growth, apoptosis, and/or differentiation. Accordingly,in one aspect, this invention provides isolated nucleic acid moleculesencoding PLTR-1 proteins or biologically active portions thereof, aswell as nucleic acid fragments suitable as primers or hybridizationprobes for the detection of PLTR-1-encoding nucleic acids.

In one embodiment, the invention features an isolated nucleic acidmolecule that includes the nucleotide sequence set forth in SEQ ID NO:19 or 21. In another embodiment, the invention features an isolatednucleic acid molecule that encodes a polypeptide including the aminoacid sequence set forth in SEQ ID NO:20.

In still other embodiments, the invention features isolated nucleic acidmolecules including nucleotide sequences that are substantiallyidentical (e.g., 75%, 79%, 80%, 81%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, or 99.9% identical) to the nucleotide sequence set forth as SEQID NO: 19 or 21. The invention further features isolated nucleic acidmolecules including at least 30, 35, 40, 45, 50, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 676, 677, 689, 690, 691, 692,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300,1350, 1400, 1450, 1500, 1550, 1562, 1600, 1610, 1660, 1700, 1750, 1800,1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2373,2374, 2375, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850,2900, 2950, 3000, 3050, 3063, 3064, 3100, 3150, 3200, 3250, 3300, 3350,3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3753, 3754, 3800, 3850,3900, 3950, 4000, 4050, 4100, 4150, 4200, 4250, 4300, 4350, 4400, 4450,4500, 4550, 4600, 4650 contiguous nucleotides of the nucleotide sequenceset forth as SEQ ID NO:19 or 21. In another embodiment, the inventionfeatures isolated nucleic acid molecules which encode a polypeptideincluding an amino acid sequence that is substantially identical (e.g.,75%, 79%, 80%, 81%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical) to the amino acid sequence set forth as SEQ ID NO:20. Alsofeatured are nucleic acid molecules which encode allelic variants of thepolypeptide having the amino acid sequence set forth as SEQ ID NO:20. Inaddition to isolated nucleic acid molecules encoding full-lengthpolypeptides, the present invention also features nucleic acid moleculeswhich encode fragments, for example, biologically active or antigenicfragments, of the full-length polypeptides of the present invention(e.g., fragments including at least 10, 15, 20, 25, 30, 25, 40, 45, 50,75, 100, 125, 150, 175, 200, 250, 300, 328, 350, 375, 400, 450, 465,500, 520, 550, 600, 650, 700, 703, 750, 800, 850, 900, 932, 950, 1000,1050, 1100, or 1150 contiguous amino acid residues of the amino acidsequence of SEQ ID NO:20). In still other embodiments, the inventionfeatures nucleic acid molecules that are complementary to, antisense to,or hybridize under stringent conditions to the isolated nucleic acidmolecules described herein.

In a related aspect, the invention provides vectors including theisolated nucleic acid molecules described herein (e.g.,PLTR-1-encoding-nucleic acid molecules). Such vectors can optionallyinclude nucleotide sequences encoding heterologous polypeptides. Alsofeatured are host cells including such vectors (e.g., host cellsincluding vectors suitable for producing PLTR-1 nucleic acid moleculesand polypeptides).

In another aspect, the invention features isolated PLTR-1 polypeptidesand/or biologically active or antigenic fragments thereof. Exemplaryembodiments feature a polypeptide including the amino acid sequence setforth as SEQ ID NO:20, a polypeptide including an amino acid sequence atleast 75%, 79%, 80%, 81%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or99.9% identical to the amino acid sequence set forth as SEQ ID NO:20, apolypeptide encoded by a nucleic acid molecule including a nucleotidesequence at least 75%, 79%, 80%, 81%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.1% 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, or 99.9% identical to the nucleotide sequence set forth as SEQ IDNO: 19 or 21. Also featured are fragments of the full-lengthpolypeptides described herein (e.g., fragments including at least 10,15, 20, 25, 30, 25, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300,328, 350, 375, 400, 450, 465, 500, 520, 550, 600, 650, 700, 703, 750,800, 850, 900, 932, 950, 1000, 1050, 1100, or 1150 contiguous amino acidresidues of the sequence set forth as SEQ ID NO:20) as well as allelicvariants of the polypeptide having the amino acid sequence set forth asSEQ ID NO:20.

The PLTR-1 polypeptides and/or biologically active or antigenicfragments thereof, are useful, for example, as reagents or targets inassays applicable to treatment and/or diagnosis of PLTR-1 associated orrelated disorders. In one embodiment, a PLTR-1 polypeptide or fragmentthereof has a PLTR-1 activity. In another embodiment, a PLTR-1polypeptide or fragment thereof has at least one or more of thefollowing domains, sites, or motifs: a transmembrane domain, anN-terminal large extramembrane domain, a C-terminal large extramembranedomain, an E1-E2 ATPases phosphorylation site, a P-type ATPase sequence1 motif, a P-type ATPase sequence 2 motif, a P-type ATPase sequence 3motif, and/or one or more phospholipid transporter specific amino acidresides, and optionally, has a PLTR-1 activity. In a related aspect, theinvention features antibodies (e.g., antibodies which specifically bindto any one of the polypeptides, as described herein) as well as fusionpolypeptides including all or a fragment of a polypeptide describedherein.

The present invention further features methods for detecting PLTR-1polypeptides and/or PLTR-1 nucleic acid molecules, such methodsfeaturing, for example, a probe, primer or antibody described herein.Also featured are kits for the detection of PLTR-1 polypeptides and/orPLTR-1 nucleic acid molecules. In a related aspect, the inventionfeatures methods for identifying compounds which bind to and/or modulatethe activity of a PLTR-1 polypeptide or PLTR-1 nucleic acid moleculedescribed herein. Also featured are methods for modulating a PLTR-1activity.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel phospholipid transporter family members, referred to herein as“Phospholipid Transporter-1” or “PLTR-1” nucleic acid and proteinmolecules. These novel molecules are capable of transportingphospholipids (e.g., aminophospholipids such as phosphatidylserine andphosphatidylethanolamine, choline phospholipids such asphosphatidylcholine and sphingomyelin, and bile acids) across cellularmembranes and, thus, play a role in or function in a variety of cellularprocesses, e.g., phospholipid transport, absorption, secretion, geneexpression, intra- or intercellular signaling, and/or cellularproliferation, growth, and/or differentiation. Thus, the PLTR-1molecules of the present invention provide novel diagnostic targets andtherapeutic agents to control PLTR-1-associated disorders, as definedherein.

The term “family” when referring to the protein and nucleic acidmolecules of the invention is intended to mean two or more proteins ornucleic acid molecules having a common structural domain or motif andhaving sufficient amino acid or nucleotide sequence homology as definedherein. Such family members can be naturally or non-naturally occurringand can be from either the same or different species. For example, afamily can contain a first protein of human origin as well as otherdistinct proteins of human origin or alternatively, can containhomologues of non-human origin, e.g., rat or mouse proteins. Members ofa family can also have common functional characteristics.

For example, the family of PLTR-1 proteins of the present inventioncomprises at least one “transmembrane domain,” preferably at least 2, 3,or 4 transmembrane domains, more preferably 5, 6, or 7 transmembranedomains, even more preferably 8 or 9 transmembrane domains, and mostpreferably, 10 transmembrane domains. As used herein, the term“transmembrane domain” includes an amino acid sequence of about 15 aminoacid residues in length which spans the plasma membrane. Morepreferably, a transmembrane domain includes about at least 20, 25, 30,35, 40, or 45 amino acid residues and spans the plasma membrane.Transmembrane domains are rich in hydrophobic residues, and typicallyhave an alpha-helical structure. In a preferred embodiment, at least50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of atransmembrane domain are hydrophobic, e.g., leucines, isoleucines,tyrosines, or tryptophans. Transmembrane domains are described in, forexample, Zagotta, W. N. et al. (1996) Annu. Rev. Neurosci. 19:235-263,the contents of which are incorporated herein by reference. Amino acidresidues 55-71, 78-94, 276-298, 320-344, 880-897, 904-924, 954-977,993-1011, 1022-1038, 1066, 1084 of the human PLTR-1 protein (SEQ IDNO:20) are predicted to comprise transmembrane domains (see FIGS. 14A-Band 15).

The family of PLTR-1 proteins of the present invention also comprises atleast one “large extramembrane domain” in the protein or correspondingnucleic acid molecule. As used herein, a “large extramembrane domain”includes a domain having greater than 20 amino acid residues that isfound between transmembrane domains, preferably on the cytoplasmic sideof the plasma membrane, and does not span or traverse the plasmamembrane. A large extramembrane domain preferably includes at least one,two, three, four or more motifs or consensus sequences characteristic ofP-type ATPases, i.e., includes one, two, three, four, or more “P-typeATPase consensus sequences or motifs”. As used herein, the phrase“P-type ATPase consensus sequences or motifs” includes any consensussequence or motif known in the art to be characteristic of P-typeATPases, including, but not limited to, the P-type ATPase sequence 1motif (as defined herein), the P-type ATPase sequence 2 motif (asdefined herein), the P-type ATPase sequence 3 motif (as defined herein),and the E1-E2 ATPases phosphorylation site (as defined herein).

In one embodiment, the family of PLTR-1 proteins of the presentinvention comprises at least one “N-terminal” large extramembrane domainin the protein or corresponding nucleic acid molecule. As used herein,an “N-terminal” large extramembrane domain is found in the N-terminal⅓^(rd) of the protein, preferably between the second and thirdtransmembrane domains of a PLTR-1 protein and includes about 60-300,80-280, 100-260, 120-240, 140-220, 160-200, or preferably, 181 aminoacid residues. In a preferred embodiment, an N-terminal largeextramembrane domain includes at least one P-type ATPase sequence 1motif (as described herein). An N-terminal large extramembrane domainwas identified in the amino acid sequence of human PLTR-1 at aboutresidues 95-275 of SEQ ID NO:20.

The family of PLTR-1 proteins of the present invention also comprises atleast one “C-terminal” large extramembrane domain in the protein orcorresponding nucleic acid molecule. As used herein, a “C-terminal”large extramembrane domain is found in the C-terminal ⅔^(rds) of theprotein, preferably between the fourth and fifth transmembrane domainsof a PLTR-1 protein and includes about 430-650, 450-630, 470-610,490-590, 510-570, 530-550, or preferably, 535 amino acid residues. In apreferred embodiment, a C-terminal large extramembrane domain includesat least one or more of the following motifs: a P-type ATPase sequence 2motif (as described herein), a P-type ATPase sequence 3 motif (asdefined herein), and/or an E1-E2 ATPases phosphorylation site (asdefined herein). A C-terminal large extramembrane domain was identifiedin the amino acid sequence of human PLTR-1 at about residues 345-879 ofSEQ ID NO:20.

In another preferred embodiment, a C-terminal large extramembrane domainincludes at least one or more of the following domains: one, two, orthree hydrolase domains and/or an Adeno_EIB_(—)19K domain. To identifythe presence of a hydrolase domain or an Adeno_E1B_(—)19K domain in aPLTR-1 family member and make the determination that a protein ofinterest has a particular profile, the amino acid sequence of theprotein is searched against a database of HMMs (e.g., the Pfam database,release 2.1) using the default parameters (available online at the PFAMwebsite, available through Washington University in St. Louis). Forexample, the hmmsf program, which is available as part of the HMMERpackage of search programs, is a family specific default program with ascore of 15 as the default threshold score for determining a hit.Alternatively, the threshold score for determining a hit can be lowered(e.g., to 8 bits). A description of the Pfam database can be found inSonhammer et al. (1997) Proteins 28(3)405-420 and a detailed descriptionof HMMs can be found, for example, in Gribskov et al. (1990) Meth.Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; andStultz et al. (1993) Protein Sci. 2:305-314, the contents of which areincorporated herein by reference. A search was performed against the HMMdatabase resulting in the identification of 3 hydrolase domains and 1Adeno_E1B_(—)19K domain in the amino acid sequence of SEQ ID NO:20. Theresults of the search are set forth below. Scores for sequence familyclassification (score includes all domains): Model Description ScoreE-value N Hydrolase haloacid dehalogenase-like hydrolase 20.9 6.5e-05 3Adeno_E1B_19K Adenovirus E1B 19K protein / small t-an  9.1 1 Parsed fordomains: Model Domain seq-f seq-t hmm-f hmm-t score E-value Hydrolase1/3 386 399 ..  1  14 [. 3.5 7.4 Adeno_E1B_19K 1/1 462 482 .. 56  76 ..9.1 0.28 Hydrolase 2/3 603 682 .. 34 104 .. 4.2 4.7 Hydrolase 3/3 762835 .. 106  184 .] 12.9  0.013            Alignments of top-scoringdomains: Hydrolase: domain 1 of 3, from 386 to 399: score 3.5, E = 7.4                   *−>ikavvFDkDGTLtd<−*                      +  ++ Dk+GTLt+     49938  386   VEYIFSDKTGTLTQ   399 Adeno_E1B_19K: domain 1 or 1,from 462 to 482: score 9.1, E = 0.28                   *−>pecpglfasLnlGytlvFqe>−*                      p+++++f++L l++t+ ++ek     49938  462   PHTHEFFRLLSLCHTVMSEEK    482 Hydrolase: domain 2 of 3,from 603 to 682: score 4.2, E = 4.7                   *−>apleevekllgrgl.gerilleggltaell......ld.evlglial                      +++e++e +++r l++   ++++++ 30  +   ++ +++  +lg+ a     49938  603   LDEEYYEEWARERRLqA-SLAQDSREDRLASiyeeveNNmMLLGATAI 648               .dklypgarealkaLkerGikvailTngdr.nae<−*               +dkl  g++e+++ L  ++ik+++lT++ +++a+      49938  649eDKLQQGVPETIALLTLANIKIWVLTGDKQeTAV   682 Hydrolase: domain 3 of 3, from762 to 835: score 12.9, E = 0.013                  *−>llealgla.lfdaivdsdevggcgpvvvgKPkpeifllalerlgvkp                     l+ al+++ +++++++++ ++  +v++ +  p  + +++e  ++     49938  762    LAHALEADmELEFLETACACK---AVICCRVTPLQKAQVVELVKKYK 805               eevgpkvlmGDginDapalaaAGvgvamgngg<−*               ++v  +l++GDg nD+ +++ A++gv +     49938  806   KAV---TLAIGDGANDVSMIKTAHIGVGISGQE   835

In another embodiment, a PLTR-1 protein includes at least one “P-typeATPase sequence 1 motif” in the protein or corresponding nucleic acidmolecule. As used herein, a “P-type ATPase sequence 1 motif” is aconserved sequence motif diagnostic for P-type ATPases (Tang, X. et al.(1996) Science 272:1495-1497; Fagan, M. J. and Saier, M. H. (1994) J.Mol. Evol. 38:57). A P-type ATPase sequence 1 motif is involved in thecoupling of ATP hydrolysis with transport (e.g., transport ofphospholipids). The consensus sequence for a P-type ATPase sequence 1motif is [DNS]-[QENR]-[SA]-[LIVSAN]-[LIV]-[TSN]-G-E-[SN] (SEQ ID NO:23).The use of amino acids in brackets indicates that the amino acid at theindicated position may be any one of the amino acids within thebrackets, e.g., [SA] indicates any of one of either S (serine) or A(alanine). In a preferred embodiment, a P-type ATPase sequence 1 motifis contained within an N-terminal large extramembrane domain. In anotherpreferred embodiment, a P-type ATPase sequence 1 motif in the PLTR-1proteins of the present invention has at least 1, 2, 3, or preferably 4amino acid resides which match the consensus sequence for a P-typeATPase sequence 1 motif. A P-type ATPase sequence 1 motif was identifiedin the amino acid sequence of human PLTR-1 at about residues 164-172 ofSEQ ID NO:20.

In another embodiment, a PLTR-1 protein includes at least one “P-typeATPase sequence 2 motif” in the protein or corresponding nucleic acidmolecule. As used herein, a “P-type ATPase sequence 2 motif” is aconserved sequence motif diagnostic for P-type ATPases (Tang, X. et al.(1996) Science 272:1495-1497; Fagan, M. J. and Saier, M. H. (1994) J.Mol. Evol. 38:57). Preferably, a P-type ATPase sequence 2 motif overlapswith and/or includes an E1-E2 ATPases phosphorylation site (as definedherein). The consensus sequence for a P-type ATPase sequence 2 motif is[LIV]-[CAML]-[STFL]-D-K-T-G-T-[LI]-T (SEQ ID NO:24). The use of aminoacids in brackets indicates that the amino acid at the indicatedposition may be any one of the amino acids within the brackets, e.g.,[LI] indicates any of one of either L (leucine) or I (isoleucine). In apreferred embodiment, a P-type ATPase sequence 2 motif is containedwithin a C-terminal large extramembrane domain. In another preferredembodiment, a P-type ATPase sequence 2 motif in the PLTR-1 proteins ofthe present invention has at least 1, 2, 3, 4, 5, 6, 7, 8, or morepreferably 9 amino acid resides which match the consensus sequence for aP-type ATPase sequence 2 motif. A P-type ATPase sequence 2 motif wasidentified in the amino acid sequence of human PLTR-1 at about residues389-398 of SEQ ID NO:20.

In yet another embodiment, a PLTR-l protein includes at least one“P-type ATPase sequence 3 motif” in the protein or corresponding nucleicacid molecule. As used herein, a “P-type ATPase sequence 3 motif” is aconserved sequence motif diagnostic for P-type ATPases (Tang, X. et al.(1996) Science 272:1495-1497; Fagan, M. J. and Saier, M. H. (1994) J.Mol. Evol. 38:57). A P-type ATPase sequence 3 motif is involved in ATPbinding. The consensus sequence for a P-type ATPase sequence 3 motif is[TIV]-G-D-G-X-N-D-[ASG]-P-[ASV]-L (SEQ ID NO:25). X indicates that theamino acid at the indicated position may be any amino acid (i.e., is notconserved). The use of amino acids in brackets indicates that the aminoacid at the indicated position may be any one of the amino acids withinthe brackets, e.g., [TIV] indicates any of one of either T (threonine),I (isoleucine), or V (valine). In a preferred embodiment, a P-typeATPase sequence 3 motif is contained within a C-terminal largeextramembrane domain. In another preferred embodiment, a P-type ATPasesequence 3 motif in the PLTR-1 proteins of the present invention has atleast 1, 2, 3, 4, 5, 6, or more preferably 7 amino acid resides(including the amino acid at the position indicated by “X”) which matchthe consensus sequence for a P-type ATPase sequence 3 motif. A P-typeATPase sequence 3 motif was identified in the amino acid sequence ofhuman PLTR-1 at about residues 812-822 of SEQ ID NO:20.

In another embodiment, a PLTR-1 protein of the present invention isidentified based on the presence of an “E1-E2 ATPases phosphorylationsite” (alternatively referred to simply as a “phosphorylation site”) inthe protein or corresponding nucleic acid molecule. An E1-E2 ATPasesphosphorylation site functions in accepting a phosphate moiety and hasthe following consensus sequence: D-K-T-G-T-[LIVM]-[TI] (SEQ ID NO:26),wherein D is phosphorylated. The use of amino acids in bracketsindicates that the amino acid at the indicated position may be any oneof the amino acids within the brackets, e.g., [TI] indicates any of oneof either T (threonine) or I (isoleucine). The E1-E2 ATPasesphosphorylation site has been assigned ProSite Accession Number PS00154.To identify the presence of an E1-E2 ATPases phosphorylation site in aPLTR-1 protein, and to make the determination that a protein of interesthas a particular profile, the amino acid sequence of the protein may besearched against a database of known protein domains (e.g., the ProSitedatabase) using the default parameters (available online through theSwiss Institute for Bioinformatics). A search was performed against theProSite database resulting in the identification of an E1-E2 ATPasesphosphorylation site in the amino acid sequence of human PLTR-1 (SEQ IDNO:20) at about residues 392-398 (see FIGS. 14A-B).

Preferably an E1-E2 ATPases phosphorylation site has a “phosphorylationsite activity,” for example, the ability to be phosphorylated; to bedephosphorylated; to regulate the E1-E2 conformational change of thephospholipid transporter in which it is contained; to regulate transportof phospholipids (e.g., aminophospholipids such as phosphatidylserineand phosphatidylethanolamine, choline phospholipids such asphosphatidylcholine and sphingomyelin, and bile acids) across a cellularmembrane by the PLTR-1 protein in which it is contained; and/or toregulate the activity (as defined herein) of the PLTR-1 protein in whichit is contained. Accordingly, identifying the presence of an “E1-E2ATPases phosphorylation site” can include isolating a fragment of aPLTR-1 molecule (e.g., a PLTR-1 polypeptide) and assaying for theability of the fragment to exhibit one of the aforementionedphosphorylation site activities.

In another embodiment, a PLTR-1 protein of the present invention mayalso be identified based on its ability to adopt an E1 conformation oran E2 conformation. As used herein, an “E1 conformation” of a PLTR-1protein includes a 3-dimensional conformation of a PLTR-1 protein whichdoes not exhibit PLTR-1 activity (e.g., the ability to transportphospholipids), as defined herein. An E1 conformation of a PLTR-1protein usually occurs when the PLTR-1 protein is unphosphorylated. Asused herein, an “E2 conformation” of a PLTR-1 protein includes a3-dimensional conformation of a PLTR-1 protein which exhibits PLTR-1activity (e.g., the ability to transport phospholipids), as definedherein. An E2 conformation of a PLTR-1 protein usually occurs when thePLTR-1 protein is phosphorylated.

In still another embodiment, a PLTR-1 protein of the present inventionis identified based on the presence of “phospholipid transporterspecific” amino acid residues. As used herein, “phospholipid transporterspecific” amino acid residues are amino acid residues specific to theclass of phospholipid transporting P-type ATPases (as defined in Tang,X. et al. (1996) Science 272:1495-1497). Phospholipid transporterspecific amino acid residues are not found in P-type ATPases whichtransport molecules which are not phospholipids (e.g., cations). Forexample, phospholipid transporter specific amino acid residues are foundat the first, second, and fifth positions of the P-type ATPase sequence1 motif. In phospholipid transporting P-type ATPases, the first positionof the P-type ATPase sequence 1 motif is preferably E (glutamic acid),the second position is preferably T (threonine), and the fifth positionis preferably L (leucine). A phospholipid transporter specific aminoacid residue is further found at the second position of the P-typeATPase sequence 2 motif. In phospholipid transporting P-type ATPases,the second position of the P-type ATPase sequence 2 motif is preferablyF (phenylalanine). Phospholipid transporter specific amino acid residuesare still further found at the first, tenth, and eleventh positions ofthe P-type ATPase sequence 3 motif. In phospholipid transporting P-typeATPases, the first position of the P-type ATPase sequence 3 motif ispreferably I (isoleucine), the tenth position is preferably M(methionine), and the eleventh position is preferably I (isoleucine).Phospholipid transporter specific amino acid residues were identified inthe amino acid sequence of human PLTR-1 (SEQ ID NO:20) at about residues164, 165, and 168 (within the P-type ATPase sequence 1 motif; see FIGS.14A-B), at about residue 390 (within the P-type ATPase sequence 2 motif;see FIGS. 14-B), and at about residues 812, 821, and 822 (within theP-type ATPase sequence 3 motif; see FIGS. 14-B).

Isolated proteins of the present invention, preferably PLTR-1 proteins,have an amino acid sequence sufficiently homologous to the amino acidsequence of SEQ ID NO:20, or are encoded by a nucleotide sequencesufficiently homologous to SEQ ID NO:19 or 21. As used herein, the term“sufficiently homologous” refers to a first amino acid or nucleotidesequence which contains a sufficient or minimum number of identical orequivalent (e.g., an amino acid residue which has a similar side chain)amino acid residues or nucleotides to a second amino acid or nucleotidesequence such that the first and second amino acid or nucleotidesequences share common structural domains or motifs and/or a commonfunctional activity. For example, amino acid or nucleotide sequenceswhich share common structural domains having at least 75%, 79%, 80%,81%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more homologyor identity across the amino acid sequences of the domains and containat least one and preferably two structural domains or motifs, aredefined herein as sufficiently homologous. Furthermore, amino acid ornucleotide sequences which share at least 75%, 79%, 80%, 81%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%,99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more homology or identity and sharea common functional activity are defined herein as sufficientlyhomologous. In a preferred embodiment, amino acid or nucleotidesequences share percent identity across the full or entire length of theamino acid or nucleotide sequence being aligned, for example, when thesequences are globally aligned (e.g., as determined by the ALIGNalgorithm as defined herein).

In a preferred embodiment, a PLTR-1 protein includes at least one ormore of the following domains, sites, or motifs: a transmembrane domain,an N-terminal large extramembrane domain, a C-terminal largeextramembrane domain, an E1-E2 ATPases phosphorylation site, a P-typeATPase sequence 1 motif, a P-type ATPase sequence 2 motif, a P-typeATPase sequence 3 motif, and/or one or more phospholipid transporterspecific amino acid resides and has an amino acid sequence at leastabout 75%, 79%, 80%, 81%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or more homologous or identical to the amino acid sequence of SEQ IDNO:20. In yet another preferred embodiment, a PLTR-1 protein includes atleast one or more of the following domains, sites, or motifs: atransmembrane domain, an N-terminal large extramembrane domain, aC-terminal large extramembrane domain, an E1-E2 ATPases phosphorylationsite, a P-type ATPase sequence 1 motif, a P-type ATPase sequence 2motif, a P-type ATPase sequence 3 motif, and/or one or more phospholipidtransporter specific amino acid resides, and is encoded by a nucleicacid molecule having a nucleotide sequence which hybridizes understringent hybridization conditions to a complement of a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:19 or 21. Inanother preferred embodiment, a PLTR-1 protein includes at least one ormore of the following domains, sites, or motifs: a transmembrane domain,an N-terminal large extramembrane domain, a C-terminal largeextramembrane domain, an E1-E2 ATPases phosphorylation site, a P-typeATPase sequence 1 motif, a P-type ATPase sequence 2 motif, a P-typeATPase sequence 3 motif, and/or one or more phospholipid transporterspecific amino acid resides, and has a PLTR-1 activity.

As used interchangeably herein, a “PLTR-1 activity”, “phospholipidtransporter activity”, “biological activity of PLTR-1”, or “functionalactivity of PLTR-1”, includes an activity exerted or mediated by aPLTR-1 protein, polypeptide or nucleic acid molecule on a PLTR-1responsive cell or on a PLTR-1 substrate, as determined in vivo or invitro, according to standard techniques. In one embodiment, a PLTR-1activity is a direct activity, such as an association with a PLTR-1target molecule. As used herein, a “target molecule” or “bindingpartner” is a molecule with which a PLTR-1 protein binds or interacts innature, such that PLTR-1-mediated function is achieved. A PLTR-1 targetmolecule can be a non-PLTR-1 molecule or a PLTR-1 protein or polypeptideof the present invention. In an exemplary embodiment, a PLTR-1 targetmolecule is a PLTR-1 substrate (e.g., a phospholipid, ATP, or anon-PLTR-1 protein). A PLTR-1 activity can also be an indirect activity,such as a cellular signaling activity mediated by interaction of thePLTR-1 protein with a PLTR-1 substrate.

In a preferred embodiment, a PLTR-1 activity is at least one of thefollowing activities: (i) interaction with a PLTR-1 substrate or targetmolecule (e.g., a phospholipid, ATP, or a non-PLTR-1 protein); (ii)transport of a PLTR-1 substrate or target molecule (e.g., anaminophospholipid such as phosphatidylserine orphosphatidylethanolamine) from one side of a cellular membrane to theother; (iii) the ability to be phosphorylated or dephosphorylated; (iv)adoption of an E1 conformation or an E2 conformation; (v) conversion ofa PLTR-1 substrate or target molecule to a product (e.g., hydrolysis ofATP); (vi) interaction with a second non-PLTR-1 protein; (vii)modulation of substrate or target molecule location (e.g., modulation ofphospholipid location within a cell and/or location with respect to acellular membrane); (viii) maintenance of aminophospholipid gradients;(ix) modulation of blood coagulation; (x) modulation of intra- orintercellular signaling and/or gene transcription (e.g., either directlyor indirectly); and/or (xi) modulation of cellular proliferation,growth, differentiation, apoptosis, absorption, or secretion.

The nucleotide sequence of the isolated human PLTR-1 cDNA and thepredicted amino acid sequence encoded by the PLTR-1 cDNA are shown inSEQ ID NOs:19 and 20, respectively.

The human PLTR-1 gene, which is approximately 4693 nucleotides inlength, encodes a protein having a molecular weight of approximately130.9 kD and which is approximately 1190 amino acid residues in length.

Various aspects of the invention are described in further detail inlater subsections.

Chapter VI. 32146 and 57259, Novel Human Transporters and Uses Thereof

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel human transporter family members, referred to herein as“transporter family members” or “TFM,” e.g., “TFM-2” and “TFM-3,”nucleic acid and polypeptide molecules. The TFM-2 and TFM-3 nucleic acidand polypeptide molecules of the present invention are useful asmodulating agents in regulating a variety of cellular processes, e.g.,cellular growth, migration, or proliferation. Accordingly, in oneaspect, this invention provides isolated nucleic acid molecules encodingTFM-2 and TFM-3 polypeptides or biologically active portions thereof, aswell as nucleic acid fragments suitable as primers or hybridizationprobes for the detection of TFM-2 and TFM-3-encoding nucleic acids.

In one embodiment, the invention features an isolated nucleic acidmolecule that includes the nucleotide sequence set forth in SEQ IDNO:27, 29, 30, or 32. In another embodiment, the invention features anisolated nucleic acid molecule that encodes a polypeptide including theamino acid sequence set forth in SEQ ID NO:28 or 31.

In still other embodiments, the invention features isolated nucleic acidmolecules including nucleotide sequences that are substantiallyidentical (e.g., 66.6%, 66.7%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%identical) to the nucleotide sequence set forth as SEQ ID NO:27, 29, 30,or 32. The invention further features isolated nucleic acid moleculesincluding at least 589, 590, 600, 650, 700, 750, 1000, 1250, 1500, 1750,or 1855 contiguous nucleotides of the nucleotide sequence set forth asSEQ ID NO:27, 29, 30, or 32. In another embodiment, the inventionfeatures isolated nucleic acid molecules which encode a polypeptideincluding an amino acid sequence that is substantially identical (e.g.,52%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical)to the amino acid sequence set forth as SEQ ID NO:28 or 31. The presentinvention also features nucleic acid molecules which encode allelicvariants of the polypeptide having the amino acid sequence set forth asSEQ ID NO:28 or 31. In addition to isolated nucleic acid moleculesencoding full-length polypeptides, the present invention also featuresnucleic acid molecules which encode fragments, for example, biologicallyactive or antigenic fragments, of the full-length polypeptides of thepresent invention (e.g., fragments including at least 157, 200, 250,300, 350, 400 or 404 contiguous amino acid residues of the amino acidsequence of SEQ ID NO:28 or 31). In still other embodiments, theinvention features nucleic acid molecules that are complementary to,antisense to, or hybridize under stringent conditions to the isolatednucleic acid molecules described herein.

In another aspect, the invention provides vectors including the isolatednucleic acid molecules described herein (e.g., TFM-2 and/orTFM-3-encoding nucleic acid molecules). Such vectors can optionallyinclude nucleotide sequences encoding heterologous polypeptides. Alsofeatured are host cells including such vectors (e.g., host cellsincluding vectors suitable for producing TFM-2 and/or TFM-3 nucleic acidmolecules and polypeptides).

In another aspect, the invention features isolated TFM-2 and TFM-3polypeptides and/or biologically active or antigenic fragments thereof.Exemplary embodiments feature a polypeptide including the amino acidsequence set forth as SEQ ID NO:28 or 31, a polypeptide including anamino acid sequence at least 52%, 55%, 60%, 65%, 70%, 75%, 80%, 85%90,95%, 98%, or 99% identical to the amino acid sequence set forth as SEQID NO:28 or 31, a polypeptide encoded by a nucleic acid moleculeincluding a nucleotide sequence at least 66.6%, 66.7%, 70%, 75%, 80%,85%, 90%, 95%, 98%, or 99% identical to the nucleotide sequence setforth as SEQ ID NO:27, 29, 30, or 32. Also featured are fragments of thefull-length polypeptides described herein (e.g., fragments including atleast 157, 200, 250, 300, 350, 400 or 404 contiguous amino acid residuesof the sequence set forth as SEQ ID NO:28 or 31) as well as allelicvariants of the polypeptide having the amino acid sequence set forth asSEQ ID NO:28 or 31.

The TFM-2 and TFM-3 polypeptides and/or biologically active or antigenicfragments thereof, are useful, for example, as reagents or targets inassays applicable to treatment and/or diagnosis of TFM-2 and TFM-3mediated or related disorders. In one embodiment, a TFM-2 and/or TFM-3polypeptide or fragment thereof, has a TFM-2 and/or TFM-3 activity. Inanother embodiment, a TFM-2 and/or TFM-3 polypeptide or fragmentthereof, includes at least one of the following domains: a transmembranedomain, a sugar transporter domain, and/or a monocarboxylate transporterdomain, and optionally, has a TFM-2 and/or a TFM-3 activity. In arelated aspect, the invention features antibodies (e.g., antibodieswhich specifically bind to any one of the polypeptides described herein)as well as fusion polypeptides including all or a fragment of apolypeptide described herein.

The present invention further features methods for detecting TFM-2 andTFM-3 polypeptides and/or TFM-2 and TFM-3 nucleic acid molecules, suchmethods featuring, for example, a probe, primer or antibody describedherein. Also featured are kits e.g., kits for the detection of TFM-2and/or TFM-3 polypeptides and/or TFM-2 and/or TFM-3 nucleic acidmolecules. In a related aspect, the invention features methods foridentifying compounds which bind to and/or modulate the activity of aTFM-2 and/or TFM-3 polypeptide or TFM-2 and/or TFM-3 nucleic acidmolecule described herein. Further featured are methods for modulating aTFM-2 and/or TFM-3 activity.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as “transporter family members” or“TFM,” e.g., “TFM-2” and. “TFM-3,” nucleic acid and polypeptidemolecules, which are novel members of the transporter family. Thesenovel molecules are capable of, for example, transporting lactate,pyruvate, branched chain oxoacids, ketone bodies, ions, proteins,sugars, and small molecules across biological membranes both within acell and between the cell and the environment and, thus, play a role inor function in a variety of cellular processes, e.g., proliferation,growth, differentiation, migration, immune responses, hormonalresponses, and inter- or intra-cellular communication.

As used herein, the term “transporter” includes a molecule which isinvolved in the movement of a biochemical molecule from one side of alipid bilayer to the other, for example, against a pre-existingconcentration gradient. Transporters are usually involved in themovement of biochemical compounds which would normally not be able tocross a membrane (e.g., a protein; an ion; a monocarboxylate; a sugar;or other small molecule, such as ATP; signaling molecules; vitamins; andcofactors). Transporter molecules are involved in the growth,development, and differentiation of cells, in the regulation of cellularhomeostasis, in the metabolism and catabolism of biochemical moleculesnecessary for energy production or storage, in intra- or inter-cellularsignaling, in metabolism or catabolism of metabolically importantbiomolecules, and in the removal of potentially harmful compounds fromthe interior of the cell. Examples of transporters includemonocarboxylate transporters, sugar transporters, GSH transporters, ATPtransporters, and fatty acid transporters. As transporters, the TFM-2and TFM-3 molecules of the present invention provide novel diagnostictargets and therapeutic agents to control transporter-associateddisorders.

As used herein, a “transporter-associated disorder” includes a disorder,disease or condition which is caused or characterized by a misregulation(e.g., downregulation or upregulation) of a transporter-mediatedactivity. Transporter-associated disorders can detrimentally affectcellular functions such as cellular proliferation, growth,differentiation, or migration, cellular regulation of homeostasis,inter- or intra-cellular communication; tissue function, such as cardiacfunction or musculoskeletal function; systemic responses in an organism,such as nervous system responses, hormonal responses (e.g., insulinresponse), or immune responses; and protection of cells from toxiccompounds (e.g., carcinogens, toxins, mutagens, and toxic byproducts ofmetabolic activity (e.g., reactive oxygen species)). Examples oftransporter-associated disorders include CNS disorders such as cognitiveand neurodegenerative disorders, examples of which include, but are notlimited to, Alzheimer's disease, dementias related to Alzheimer'sdisease (such as Pick's disease), Parkinson's and other Lewy diffusebody diseases, senile dementia, Huntington's disease, Gilles de laTourette's syndrome, multiple sclerosis, amyotrophic lateral sclerosis,progressive supranuclear palsy, epilepsy, and Creutzfeldt-Jakob disease;autonomic function disorders such as hypertension and sleep disorders,and neuropsychiatric disorders, such as depression, schizophrenia,schizoaffective disorder, korsakoff's psychosis, mania, anxietydisorders, or phobic disorders; learning or memory disorders, e.g.,amnesia or age-related memory loss, attention deficit disorder,dysthymic disorder, major depressive disorder, mania,obsessive-compulsive disorder, psychoactive substance use disorders,anxiety, phobias, panic disorder, as well as bipolar affective disorder,e.g., severe bipolar affective (mood) disorder (BP-1), and bipolaraffective neurological disorders, e.g., migraine and obesity. FurtherCNS-related disorders include, for example, those listed in the AmericanPsychiatric Association's Diagnostic and Statistical manual of MentalDisorders (DSM), the most current version of which is incorporatedherein by reference in its entirety.

Further examples of transporter-associated disorders includecardiac-related disorders. Cardiovascular system disorders in which theTFM-2 and TFM-3 molecules of the invention may be directly or indirectlyinvolved include arteriosclerosis, ischemia reperfusion injury,restenosis, arterial inflammation, vascular wall remodeling, ventricularremodeling, rapid ventricular pacing, coronary microembolism,tachycardia, bradycardia, pressure overload, aortic bending, coronaryartery ligation, vascular heart disease, atrial fibrilation, Jervellsyndrome, Lange-Nielsen syndrome, long-QT syndrome, congestive heartfailure, sinus node dysfunction, angina, heart failure, hypertension,atrial fibrillation, atrial flutter, dilated cardiomyopathy, idiopathiccardiomyopathy, myocardial infarction, coronary artery disease, coronaryartery spasm, and arrhythmia. TFM-2 and TFM-3-mediated or relateddisorders also include disorders of the musculoskeletal system such asparalysis and muscle weakness, e.g., ataxia, myotonia, and myokymia.

Transporter-associated disorders also include cellular proliferation,growth, differentiation, or migration disorders. Cellular proliferation,growth, differentiation, or migration disorders include those disordersthat affect cell proliferation, growth, differentiation, or migrationprocesses. As used herein, a “cellular proliferation, growth,differentiation, or migration process” is a process by which a cellincreases in number, size or content, by which a cell develops aspecialized set of characteristics which differ from that of othercells, or by which a cell moves closer to or further from a particularlocation or stimulus. The TFM-2 and TFM-3 molecules of the presentinvention are involved in signal transduction mechanisms, which areknown to be involved in cellular growth, differentiation, and migrationprocesses. Thus, the TFM-2 and TFM-3 molecules may modulate cellulargrowth, differentiation, or migration, and may play a role in disorderscharacterized by aberrantly regulated growth, differentiation, ormigration. Such disorders include cancer, e.g., carcinoma, sarcoma, orleukemia; tumor angiogenesis and metastasis; skeletal dysplasia; hepaticdisorders; and hematopoietic and/or myeloproliferative disorders.

Transporter-associated disorders also include hormonal disorders, suchas conditions or diseases in which the production and/or regulation ofhormones in an organism is aberrant. Examples of such disorders anddiseases include type I and type II diabetes mellitus, pituitarydisorders (e.g., growth disorders), thyroid disorders (e.g.,hypothyroidism or hyperthyroidism), and reproductive or fertilitydisorders (e.g., disorders which affect the organs of the reproductivesystem, e.g., the prostate gland, the uterus, or the vagina; disorderswhich involve an imbalance in the levels of a reproductive hormone in asubject; disorders affecting the ability of a subject to reproduce; anddisorders affecting secondary sex characteristic development, e.g.,adrenal hyperplasia).

Transporter-associated disorders also include immune disorders, such asautoimmune disorders or immune deficiency disorders, e.g., congenitalX-linked infantile hypogammaglobulinemia, transienthypogammaglobulinemia, common variable immunodeficiency, selective IgAdeficiency, chronic mucocutaneous candidiasis, or severe combinedimmunodeficiency.

Transporter-associated disorders also include disorders associated withsugar homeostasis, such as obesity, anorexia, hypoglycemia, glycogenstorage disease (Von Gierke disease), type I glycogenosis, seasonalaffective disorder, and cluster B personality disorders.

Transporter-associated disorders also include disorders affectingtissues in which TFM-2 and TFM-3 protein is expressed.

As used herein, a “transporter-mediated activity” includes an activityof a transporter which involves the facilitated movement of one or moremolecules, e.g., biological molecules, from one side of a biologicalmembrane to the other. Transporter-mediated activities include theimport or export across internal or external cellular membranes ofbiochemical molecules necessary for energy production or storage; intra-or inter-cellular signaling; metabolism or catabolism of metabolicallyimportant biomolecules; and removal of potentially harmful compoundsfrom the cell.

The term “family” when referring to the polypeptide and nucleic acidmolecules of the invention is intended to mean two or more polypeptidesor nucleic acid molecules having a common structural domain or motif andhaving sufficient amino acid or nucleotide sequence homology as definedherein. Such family members can be naturally or non-naturally occurringand can be from either the same or different species. For example, afamily can contain a first polypeptide of human origin, as well asother, distinct polypeptides of human origin or alternatively, cancontain homologues of non-human origin, e.g., mouse or monkeypolypeptides. Members of a family may also have common functionalcharacteristics.

For example, the family of TFM-2 and TFM-3 polypeptides comprise atleast one “transmembrane domain” and preferably eight, nine, or tentransmembrane domains. As used herein, the term “transmembrane domain”includes an amino acid sequence of about 15-45 amino acid residues inlength which spans the plasma membrane. More preferably, a transmembranedomain includes about at least 15, 20, 25, 30, 35, 40, or 45 amino acidresidues and spans the plasma membrane. Transmembrane domains are richin hydrophobic residues, and typically have an alpha-helical structure.In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or moreof the amino acids of a transmembrane domain are hydrophobic, e.g.,leucines, isoleucines, alanines, valines, phenylalanines, prolines ormethionines. Transmembrane domains are described in, for example,Zagotta W. N. et al, (1996) Annual Rev. Neurosci. 19: 235-263, thecontents of which are incorporated herein by reference. A MEMSATanalysis and a structural, hydrophobicity, and antigenicity analysisalso resulted in the identification of ten transmembrane domains in theamino acid sequence of human TFM-2 (SEQ ID NO:28) at about residues22-42, 49-69, 76-98, 105-128, 167-186, 207-223, 236-253, 261-285,296-318, and 327-349 as set forth in FIGS. 16 and 17. A MEMSAT analysisand a structural, hydrophobicity, and antigenicity analysis resulted inthe identification of nine transmembrane domains in the amino acidsequence of human TFM-3 (SEQ ID NO:31) at about residues 7-23, 34-57,66-82, 150-168, 188-206, 213-237, 255-279, 288-308, and 321-337 as setforth in FIGS. 18 and 19.

Accordingly, TFM-2 and/or TFM-3 polypeptides having at least 50-60%homology, preferably about 60-70%, more preferably about 70-80%, orabout 80-90% homology with a transmembrane domain of human TFM-2 and/orTFM-3 are within the scope of the invention.

In one embodiment, a TFM molecule of the present invention, e.g., TFM-2,is identified based on the presence within the molecule of at least one“monocarboxylate transporter domain.” As used herein, the term“monocarboxylate transporter domain” includes a protein domain having atleast about 250-500 amino acid residues, a bit score of at least 20 whencompared against a monocarboxylate transporter domain Hidden MarkovModel, and a monocarboxylate transporter mediated activity. Preferably,a monocarboxylate transporter domain includes a protein domain having anamino acid sequence of about 300-400, 300-350, or more preferably, about330 amino acid residues, a bit score of at least 35, and amonocarboxylate transporter mediated activity. To identify the presenceof a monocarboxylate transporter domain in a TFM-2 protein, and make thedetermination that a protein of interest has a particular profile, theamino acid sequence of the protein may be searched against a database ofknown protein domains (e.g., the PFAM HMM database). A PFAMmonocarboxylate transporter domain has been assigned the PFAM AccessionPF01587. A search was performed against the PFAM HMM database resultingin the identification of a monocarboxylate transporter domain in theamino acid sequence of human TFM-2 (SEQ ID NO:28) at about residues1-332 of SEQ ID NO:28.

As used herein, a “monocarboxylate transporter mediated activity”includes the ability to mediate the transport of a variety ofmonocarboxylates (e.g., lactate, pyruvate, branched chain oxoacids,and/or ketone bodies) across a biological membrane (e.g., a red bloodcell membrane, a heart cell membrane, a brain cell membrane, a skeletalmuscle cell membrane, a liver cell membrane, a kidney cell membrane,and/or a tumor cell membrane. Accordingly, identifying the presence of a“monocarboxylate transporter domain” can include isolating a fragment ofa TFM-2 molecule (e.g., a TFM-2 polypeptide) and assaying for theability of the fragment to exhibit one of the aforementionedmonocarboxylate transporter mediated activities.

In another embodiment, members of the TFM family of proteins, e.g.,TFM-3, include at least one “sugar transporter domain” in the protein orcorresponding nucleic acid molecule. As used herein, the term “sugartransporter domain” includes a protein domain having at least about250-500 amino acid residues and a sugar transporter mediated activity.Preferably, a sugar transporter domain includes a polypeptide having anamino acid sequence of about 300-400, 300-350, or more preferably, about353 amino acid residues, and a sugar transporter mediated activity. Toidentify the presence of a sugar transporter domain in a TFM-3 protein,and make the determination that a protein of interest has a particularprofile, the amino acid sequence of the protein may be searched againsta database of known protein domains (e.g., the PFAM HMM database). APFAM sugar transporter domain has been assigned the PFAM AccessionPF00083. A search was performed against the PFAM HMM database resultingin the identification of a sugar transporter domain in the amino acidsequence of human TFM-3 (SEQ ID NO:31) at about residues 1-353 of SEQ IDNO:31.

As used herein, a “sugar transporter mediated activity” includes theability to bind a monosaccharide, such as D-glucose, D-fructose, and/orD-galactose; the ability to transport a monosaccharide such asD-glucose, D-fructose, and/or D-galactose, across a cell membrane (e.g.,a liver cell membrane, fat cell membrane, muscle cell membrane, and/orblood cell membrane, such as an erythrocyte membrane); and the abilityto modulate sugar homeostasis in a cell. Accordingly, identifying thepresence of a “sugar transporter domain” can include isolating afragment of a TFM-3 molecule (e.g., a TFM-3 polypeptide) and assayingfor the ability of the fragment to exhibit one of the aforementionedsugar transporter mediated activities.

A description of the Pfam database can be found in Sonhammer et al.(1997) Proteins 28:405-420 and a detailed description of HMMs can befound, for example, in Gribskov et al. (1990) Meth. Enzymol.183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; andStultz et al. (1993) Protein Sci. 2:305-314, the contents of which areincorporated herein by reference.

In a preferred embodiment, the TFM-2 and TFM-3 molecules of theinvention include at least one, preferably two, even more preferablyeight, nine or ten transmembrane domain(s), and/or at least onemonocarboxylate transporter domain, and/or at least one sugartransporter domain.

Isolated polypeptides of the present invention, preferably TFM-2 and/orTFM-3 polypeptides, have an amino acid sequence sufficiently identicalto the amino acid sequence of SEQ ID NO:28 or 31 or are encoded by anucleotide sequence sufficiently identical to SEQ ID NO:27, 29, 30, or32. As used herein, the term “sufficiently identical” refers to a firstamino acid or nucleotide sequence which contains a sufficient or minimumnumber of identical or equivalent (e.g., an amino acid residue which hasa similar side chain) amino acid residues or nucleotides to a secondamino acid or nucleotide sequence such that the first and second aminoacid or nucleotide sequences share common structural domains or motifsand/or a common functional activity. For example, amino acid ornucleotide sequences which share common structural domains having atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%,98%, 99% or more homology or identity across the amino acid sequences ofthe domains and contain at least one and preferably two structuraldomains or motifs, are defined herein as sufficiently identical.Furthermore, amino acid or nucleotide sequences which share at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%,99% or more homology or identity and share a common functional activityare defined herein as sufficiently identical.

In a preferred embodiment, a TFM-2 and/or a TFM-3 polypeptide includesat least one or more of the following domains: a transmembrane domain,and/or a monocarboxylate transporter domain, and/or a sugar transporterdomain, and has an amino acid sequence at least about 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or morehomologous or identical to the amino acid sequence of SEQ ID NO:28 or31. In yet another preferred embodiment, a TFM-2 and/or a TFM-3polypeptide includes at least one or more of the following domains: atransmembrane domain, and/or a monocarboxylate transporter domain,and/or a sugar transporter domain, and is encoded by a nucleic acidmolecule having a nucleotide sequence which hybridizes under stringenthybridization conditions to a complement of a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:27, 29, 30, or 32. Inanother preferred embodiment, a TFM-2 and/or a TFM-3 polypeptideincludes at least one or more of the following domains: a transmembranedomain, and/or a monocarboxylate transporter domain, and/or a sugartransporter domain, and has a TFM-2 and/or TFM-3 activity.

As used interchangeably herein, a “TFM-2 activity,” “TFM-3 activity,”“biological activity of TFM-2,” “biological activity of TFM-3,”“functional activity of TFM-2,” or “functional activity of TFM-3” refersto an activity exerted by a TFM-2 and/or a TFM-3 protein, polypeptide ornucleic acid molecule on a TFM-2 and/or a TFM-3 responsive cell ortissue, or on a TFM-2 and/or a TFM-3 protein substrate, as determined invivo, or in vitro, according to standard techniques. In one embodiment,a TFM-2 and/or a TFM-3 activity is a direct activity, such as anassociation with a TFM-2 and/or a TFM-3-target molecule. As used herein,a “target molecule” or “binding partner” is a molecule with which aTFM-2 and/or a TFM-3 protein binds or interacts in nature, such thatTFM-2 and/or TFM-3-mediated function is achieved. A TFM-2 and/or a TFM-3target molecule can be a non-TFM-2 and/or a non-TFM-3 molecule or aTFM-2 and/or a TFM-3 protein or polypeptide of the present invention(e.g., a molecule to be transported, e.g., a monocarboxylate and/or amonosaccharide). In an exemplary embodiment, a TFM-2 and/or a TFM-3target molecule is a TFM-2 and/or a TFM-3 ligand (e.g., a proton, anenergy molecule, a metabolite, a monocarboxylate, a monosaccharide or anion). Alternatively, a TFM-2 and/or a TFM-3 activity is an indirectactivity, such as a cellular signaling activity mediated by interactionof the TFM-2 and/or a TFM-3 protein with a TFM-2 and/or a TFM-3 ligand.The biological activities of TFM-2 and TFM-3 are described herein. Forexample, the TFM-2 and/or TFM-3 proteins of the present invention canhave one or more of the following activities: 1) modulate the import andexport of molecules, e.g., hormones, ions, cytokines, neurotransmitters,monocarboxylates, monosaccharides, and metabolites, from cells, 2)modulate intra- or inter-cellular signaling, 3) modulate removal ofpotentially harmful compounds from the cell, or facilitate thecompartmentalization of these molecules into a sequesteredintra-cellular space (e.g., the peroxisome), and 4) modulate transportof biological molecules across membranes, e.g., the plasma membrane, orthe membrane of the mitochondrion, the peroxisome, the lysosome, theendoplasmic reticulum, the nucleus, or the vacuole.

The nucleotide sequence of the isolated human TFM-2 and TFM-3 cDNA andthe predicted amino acid sequence of the human TFM-2 and TFM-3polypeptides are shown in SEQ ID NOs:27, 28 and 30, 31, respectively.

The human TFM-2 gene, which is approximately 3524 nucleotides in length,encodes a polypeptide which is approximately 392 amino acid residues inlength. The human TFM-3 gene, which is approximately 1855 nucleotides inlength, encodes a polypeptide which is approximately 405 amino acidresidues in length.

Various aspects of the invention are described in further detail in thefollowing subsections:

Chapter VII. 67118, 67067, and 62092, Human Proteins and Methods of UseThereof

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel human phospholipid transporter family members, referred to hereinas “67118 and 67067” nucleic acid and polypeptide molecules. The 67118and 67067 nucleic acid and polypeptide molecules of the presentinvention are useful as modulating agents in regulating a variety ofcellular processes, e.g., phospholipid transport (e.g.,aminophospholipid transport), absorption, secretion, gene expression,intra- or inter-cellular signaling, and/or cellular proliferation,growth, apoptosis, and/or differentiation. Accordingly, in one aspect,this invention provides isolated nucleic acid molecules encoding 67118and 67067 polypeptides or biologically active portions thereof, as wellas nucleic acid fragments suitable as primers or hybridization probesfor the detection of 67118 and 67067-encoding nucleic acids.

The present invention is also based, at least in part, on the discoveryof novel histidine triad family members, referred to herein as “62092”nucleic acid and protein molecules. The 62092 nucleic acid and proteinmolecules of the present invention are useful as modulating agents inregulating a variety of cellular processes, e.g., gene expression,intra- or intercellular signaling, cellular proliferation, growth,differentiation, and/or apoptosis, and/or sensing of cellular stresssignals. Accordingly, in one aspect, this invention provides isolatednucleic acid molecules encoding 62092 proteins or biologically activeportions thereof, as well as nucleic acid fragments suitable as primersor hybridization probes for the detection of 62092-encoding nucleicacids.

In one embodiment, the invention features an isolated nucleic acidmolecule that includes the nucleotide sequence set forth in SEQ IDNO:33, 35, 36, 38, 39, or 41. In another embodiment, the inventionfeatures an isolated nucleic acid molecule that encodes a polypeptideincluding the amino acid sequence set forth in SEQ ID NO:34, 37, or 40.

In still other embodiments, the invention features isolated nucleic acidmolecules including nucleotide sequences that are substantiallyidentical (e.g., 60% identical) to the nucleotide sequence set forth asSEQ ID NO:33, 35, 36, 38, 39, or 41. The invention further featuresisolated nucleic acid molecules including at least 50 contiguousnucleotides of the nucleotide sequence set forth as SEQ ID NO:33, 35,36, 38, 39, or 41. In another embodiment, the invention featuresisolated nucleic acid molecules which encode a polypeptide including anamino acid sequence that is substantially identical (e.g., 60%identical) to the amino acid sequence set forth as SEQ ID NO:34, 37, or40. The present invention also features nucleic acid molecules whichencode allelic variants of the polypeptide having the amino acidsequence set forth as SEQ ID NO:34, 37, or 40. In addition to isolatednucleic acid molecules encoding full-length polypeptides, the presentinvention also features nucleic acid molecules which encode fragments,for example, biologically active or antigenic fragments, of thefull-length polypeptides of the present invention (e.g., fragmentsincluding at least 10 contiguous amino acid residues of the amino acidsequence of SEQ ID NO:34, 37, or 40). In still other embodiments, theinvention features nucleic acid molecules that are complementary to,antisense to, or hybridize under stringent conditions to the isolatednucleic acid molecules described herein.

In another aspect, the invention provides vectors including the isolatednucleic acid molecules described herein (e.g., 67118, 67067, and/or62092-encoding nucleic acid molecules). Such vectors can optionallyinclude nucleotide sequences encoding heterologous polypeptides. Alsofeatured are host cells including such vectors (e.g., host cellsincluding vectors suitable for producing 67118, 67067, and/or 62092nucleic acid molecules and polypeptides).

In another aspect, the invention features isolated 67118, 67067, and/or62092 polypeptides and/or biologically active or antigenic fragmentsthereof. Exemplary embodiments feature a polypeptide including the aminoacid sequence set forth as SEQ ID NO:34, 37, or 40, a polypeptideincluding an amino acid sequence at least 60% identical to the aminoacid sequence set forth as SEQ ID NO:34, 37, or 40, a polypeptideencoded by a nucleic acid molecule including a nucleotide sequence atleast 60% identical to the nucleotide sequence set forth as SEQ IDNO:33, 35, 36, 38, 39, or 41. Also featured are fragments of thefull-length polypeptides described herein (e.g., fragments including atleast 10 contiguous amino acid residues of the sequence set forth as SEQID NO:34, 37, or 40) as well as allelic variants of the polypeptidehaving the amino acid sequence set forth as SEQ ID NO:34, 37, or 40.

The 67118, 67067, and/or 62092 polypeptides and/or biologically activeor antigenic fragments thereof, are useful, for example, as reagents ortargets in assays applicable to treatment and/or diagnosis of 67118,67067, and/or 62092 associated or related disorders. In one embodiment,a 67118, 67067, and/or 62092 polypeptide or fragment thereof, has a67118, 67067, and/or 62092 activity.

In another embodiment, a 67118 or 67067 polypeptide or fragment thereofincludes at least one of the following domains, sites, or motifs: atransmembrane domain, an N-terminal large extramembrane domain, aC-terminal large extramembrane domain, an E1-E2 ATPases phosphorylationsite, a P-type ATPase sequence 1 motif, a P-type ATPase sequence 2motif, a P-type ATPase sequence 3 motif, and/or one or more phospholipidtransporter specific amino acid resides, and optionally, has a 67118and/or a 67067 activity. In yet another embodiment, a 62092 polypeptideor fragment thereof has at least one or more of the following domains ormotifs: a signal peptide, a HIT family domain, and/or a HIT familysignature motif, and optionally, has a 62092 activity.

In a related aspect, the invention features antibodies (e.g., antibodieswhich specifically bind to any one of the polypeptides described herein)as well as fusion polypeptides including all or a fragment of apolypeptide described herein.

The present invention further features methods for detecting 67118,67067, and/or 62092 polypeptides and/or 67118, 67067, and/or 62092nucleic acid molecules, such methods featuring, for example, a probe,primer or antibody described herein. Also featured are kits, e.g., kitsfor the detection of 67118, 67067, and/or 62092 polypeptides and/or67118, 67067, and/or 62092 nucleic acid molecules. In a related aspect,the invention features methods for identifying compounds which bind toand/or modulate the activity of a 67118, 67067, and/or 62092 polypeptideor 67118, 67067, and/or 62092 nucleic acid molecule described herein.Further featured are methods for modulating a 67118, 67067, and/or 62092activity.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as “67118” and “67067” nucleic acidand polypeptide molecules, which are novel members of the phospholipidtransporter family. These novel molecules are capable of, for example,transporting phospholipids (e.g., aminophospholipids such asphosphatidylserine and phosphatidylethanolamine, choline phospholipidssuch as phosphatidylcholine and sphingomyelin, and bile acids) acrosscellular membranes and, thus, play a role in or function in a variety ofcellular processes, e.g., phospholipid transport, absorption, secretion,gene expression, intra- or inter-cellular signaling, and/or cellularproliferation, growth, and/or differentiation.

The present invention is also based, at least in part, on the discoveryof novel histidine triad family members, referred to herein as “62092”nucleic acid and protein molecules. These novel molecules are capable ofbinding nucleotides (e.g., purine mononucleotides and/or dinucleosidepolyphosphates) and, thus, play a role in or function in a variety ofcellular processes, e.g., gene expression, intra- or intercellularsignaling, cellular proliferation, growth, differentiation, and/orapoptosis, and/or sensing of cellular stress signals. Thus, the 62092molecules of the present invention provide novel diagnostic targets andtherapeutic agents to control 62092-associated disorders, as definedherein.

The term “family” when referring to the protein and nucleic acidmolecules of the invention is intended to mean two or more proteins ornucleic acid molecules having a common structural domain or motif andhaving sufficient amino acid or nucleotide sequence homology as definedherein. Such family members can be naturally or non-naturally occurringand can be from either the same or different species. For example, afamily can contain a first protein of human origin as well as otherdistinct proteins of human origin or alternatively, can containhomologues of non-human origin, e.g., rat or mouse proteins. Members ofa family can also have common functional characteristics.

For example, the family of 67118 and 67067 polypeptides comprise atleast one “transmembrane domain” and preferably eight, nine, or tentransmembrane domains. As used herein, the term “transmembrane domain”includes an amino acid sequence of about 15-45 amino acid residues inlength which spans the plasma membrane. More preferably, a transmembranedomain includes about at least 15, 20, 25, 30, 35, 40, or 45 amino acidresidues and spans the plasma membrane. Transmembrane domains are richin hydrophobic residues, and typically have an alpha-helical structure.In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or moreof the amino acids of a transmembrane domain are hydrophobic, e.g.,leucines, isoleucines, alanines, valines, phenylalanines, prolines ormethionines. Transmembrane domains are described in, for example,Zagotta W. N. et al, (1996) Annual Rev. Neurosci. 19: 235-263, thecontents of which are incorporated herein by reference. A MEMSATanalysis and a structural, hydrophobicity, and antigenicity analysisalso resulted in the identification of ten transmembrane domains in theamino acid sequence of human 67118 (SEQ ID NO:34) at about residues71-87, 94-110, 295-314, 349-368, 891-907, 915-935, 964-987, 1002-1018,1033-1057, and 1064-1088 as set forth in FIG. 20. A MEMSAT analysis anda structural, hydrophobicity, and antigenicity analysis resulted in theidentification of ten transmembrane domains in the amino acid sequenceof human 67067 (SEQ ID NO:37) at about residues 65-82, 89-105, 287-304,366-388, 1239-1259, 1322-1343, 1274-1292, 1351-1368, 1377-1399,1425-1446 as set forth in FIG. 22.

The family of 67118 and/or 67067 proteins of the present invention alsocomprise at least one “extramembrane domain” in the protein orcorresponding nucleic acid molecule. As used herein, an “extramembranedomain” includes a domain having greater than 20 amino acid residuesthat is found between transmembrane domains, preferably on thecytoplasmic side of the plasma membrane, and does not span or traversethe plasma membrane. An extramembrane domain preferably includes atleast one, two, three, four or more motifs or consensus sequencescharacteristic of P-type ATPases, i.e., includes one, two, three, four,or more “P-type ATPase consensus sequences or motifs”. As used herein,the phrase “P-type ATPase consensus sequences or motifs” includes anyconsensus sequence or motif known in the art to be characteristic ofP-type ATPases, including, but not limited to, the P-type ATPasesequence 1 motif (as defined herein), the P-type ATPase sequence 2 motif(as defined herein), the P-type ATPase sequence 3 motif (as definedherein), and the E1-E2 ATPases phosphorylation site (as defined herein).

In one embodiment, the family of 67118 and 67067 proteins of the presentinvention comprises at least one “N-terminal” large extramembrane domainin the protein or corresponding nucleic acid molecule. As used herein,an “N-terminal” large extramembrane domain is found in the N-terminal⅓^(rd) of the protein, preferably between the second and thirdtransmembrane domains of a 67118 or 67067 protein and includes about60-300, 80-280, 100-260, 120-240, 140-220, 160-200, or preferably,181 or183 amino acid residues. In a preferred embodiment, an N-terminal largeextramembrane domain includes at least one P-type ATPase sequence 1motif (as described herein). An N-terminal large extramembrane domainwas identified in the amino acid sequence of human 67118 at aboutresidues 111-294 of SEQ ID NO:34. An N-terminal large extramembranedomain was identified in the amino acid sequence of human 67067 at aboutresidues 105-286 of SEQ ID NO:37.

The family of 67118 and 67067 proteins of the present invention alsocomprises at least one “C-terminal” large extramembrane domain in theprotein or corresponding nucleic acid molecule. As used herein, a“C-terminal” large extramembrane domain is found in the C-terminal⅔^(rds) of the protein, preferably between the fourth and fifthtransmembrane domains of a PLTR protein and includes about 370-850,400-820, 430-790, 460-760, 430-730, 460-700, 430-670, 460-640, 430-610,490-580, 510-550, or preferably, 521 or 849 amino acid residues. In apreferred embodiment, a C-terminal large extramembrane domain includesat least one or more of the following motifs: a P-type ATPase sequence 2motif (as described herein), a P-type ATPase sequence 3 motif (asdefined herein), and/or an E1-E2 ATPases phosphorylation site (asdefined herein). A C-terminal large extramembrane domain was identifiedin the amino acid sequence of human 67118 at about residues 369-890 ofSEQ ID NO:34. A C-terminal large extramembrane domain was identified inthe amino acid sequence of human 67067 at about residues 389-1238 of SEQID NO:37.

In another embodiment, a 67118 or 67067 protein extramembrane domain ischaracterized by at least one “P-type ATPase sequence 1 motif” in theprotein or corresponding nucleic acid sequence. As used herein, a“P-type ATPase sequence 1 motif” is a conserved sequence motifdiagnostic for P-type ATPases (Tang, X. et al. (1996) Science272:1495-1497; Fagan, M. J. and Saier, M. H. (1994) J. Mol. Evol.38:57). Amino acid residues of the P-type ATPase sequence 1 motif areinvolved in the coupling of ATP hydrolysis with transport (e.g.,transport of phospholipids). The consensus sequence for a P-type ATPasesequence 1 motif is [DNS]-[QENR]-[SA]-[LIVSAN]-[LIV]-[TSN]-G-E-[SN] (SEQID NO:42). The use of amino acids in brackets indicates that the aminoacid at the indicated position may be any one of the amino acids withinthe brackets, e.g., [SA] indicates any of one of either S (serine) or A(alanine). In a preferred embodiment, a P-type ATPase sequence 1 motifis contained within an N-terminal large extramembrane domain. In anotherpreferred embodiment, a P-type ATPase sequence I motif in the 67118,67067, and/or 62092 proteins of the present invention has at least 1, 2,3, or preferably 4 amino acid resides which match the consensus sequencefor a P-type ATPase sequence 1 motif. A P-type ATPase sequence 1. motifwas identified in the amino acid sequence of human 67118 at aboutresidues 179-187 of SEQ ID NO:34. A P-type ATPase sequence 1 motif wasidentified in the amino acid sequence of human 67067 at about residues175-183 of SEQ ID NO:37.

In another embodiment, a 67118 or 67067 protein extramembrane domain ischaracterized by at least one “P-type ATPase sequence 2 motif” in theprotein or corresponding nucleic acid sequence. As used herein, a“P-type ATPase sequence 2 motif” is a conserved sequence motifdiagnostic for P-type ATPases (Tang, X. et al. (1996) Science272:1495-1497; Fagan, M. J. and Saier, M. H. (1994) J. Mol. Evol.38:57). Preferably, a P-type ATPase sequence 2 motif overlaps withand/or includes an E1-E2 ATPases phosphorylation site (as definedherein). The consensus sequence for a P-type ATPase sequence 2 motif is[LIV]-[CAML]-[STFL]-D-K-T-G-T-[LI]-T (SEQ ID NO:43). The use of aminoacids in brackets indicates that the amino acid at the indicatedposition may be any one of the amino acids within the brackets, e.g.,[LI] indicates any of one of either L (leucine) or I (isoleucine). In apreferred embodiment, a P-type ATPase sequence 2 motif is containedwithin a C-terminal large extramembrane domain. In another preferredembodiment, a P-type ATPase sequence 2 motif in the PLTR proteins of thepresent invention has at least 1, 2, 3, 4, 5, 6, 7, 8, or morepreferably 9 amino acid resides which match the consensus sequence for aP-type ATPase sequence 2 motif. A P-type ATPase sequence 2 motif wasidentified in the amino acid sequence of human 67118 at about residues411-420 of SEQ ID NO:34. A P-type ATPase sequence 2 motif was identifiedin the amino acid sequence of human 67067 at about residues 431-440 ofSEQ ID NO:37.

In yet another embodiment, a 67118 or 67067 protein extramembrane domainis characterized by at least one “P-type ATPase sequence 3 motif” in theprotein or corresponding nucleic acid sequence. As used herein, a“P-type ATPase sequence 3 motif” is a conserved sequence motifdiagnostic for P-type ATPases (Tang, X. et al. (1996) Science272:1495-1497; Fagan, M. J. and Saier, M. H. (1994) J. Mol. Evol.38:57). Amino acid residues of the P-type ATPase sequence 3 motif areinvolved in ATP binding. The consensus sequence for a P-type ATPasesequence 3 motif is [TIV]-G-D-G-X-N-D-[ASG]-P-[ASV]-L (SEQ ID NO:44). Xindicates that the amino acid at the indicated position may be any aminoacid (i.e., is not conserved). The use of amino acids in bracketsindicates that the amino acid at the indicated position may be any oneof the amino acids within the brackets, e.g., [TIV] indicates any of oneof either T (threonine), I (isoleucine), or V (valine). In a preferredembodiment, a P-type ATPase sequence 3 motif is contained within aC-terminal large extramembrane domain. In another preferred embodiment,a P-type ATPase sequence 3 motif in the 67118 or 67067 proteins of thepresent invention has at least 1, 2, 3, 4, 5, 6, or more preferably 7amino acid resides (including the amino acid at the position indicatedby “X”) which match the consensus sequence for a P-type ATPase sequence3 motif. A P-type ATPase sequence 3 motif was identified in the aminoacid sequence of human 67118 at about residues 823-833 of SEQ ID NO:34.A P-type ATPase sequence 3 motif was identified in the amino acidsequence of human 67067 at about residues 1180-1190 of SEQ ID NO:37.

In another embodiment, a 67118 or 67067 protein of the present inventionis identified based on the presence of an “E1-E2 ATPases phosphorylationsite” (alternatively referred to simply as a “phosphorylation site”) inthe protein or corresponding nucleic acid molecule. An E1-E2 ATPasesphosphorylation site functions in accepting a phosphate moiety and hasthe amino acid sequence DKTGT (amino acid residues 1-5 of SEQ ID NO:45),and can be included within the E1-E2 ATPase phosphorylation siteconsensus sequence: D-K-T-G-T-[LIVM]-[TI] (SEQ ID NO:45), wherein D isphosphorylated. The use of amino acids in brackets indicates that theamino acid at the indicated position may be any one of the amino acidswithin the brackets, e.g., [TI] indicates any of one of either T(threonine) or I (isoleucine). The E1-E2 ATPases phosphorylation siteconsensus sequence has been assigned ProSite Accession Number PS00154.To identify the presence of an E1-E2 ATPases phosphorylation siteconsensus sequence in a 67118 or 67067 protein, and to make thedetermination that a protein of interest has a particular profile, theamino acid sequence of the protein may be searched against a database ofknown protein motifs (e.g., the ProSite database) using the defaultparameters (available on the Internet at the Prosite website). A searchwas performed against the ProSite database resulting in theidentification of an E1-E2 ATPases phosphorylation site consensussequence in the amino acid sequence of human 67118 (SEQ ID NO:34) atabout residues 414-420 (see FIGS. 21A-B). A search was performed againstthe ProSite database resulting in the identification of an E1-E2 ATPasesphosphorylation site consensus sequence in the amino acid sequence ofhuman 67067 (SEQ ID NO:37) at about residues 434-440 (see FIGS. 23A-B).

Preferably an E1-E2 ATPases phosphorylation site has a “phosphorylationsite activity,” for example, the ability to be phosphorylated; to bedephosphorylated; to regulate the E1-E2 conformational change of thephospholipid transporter in which it is contained; to regulate transportof phospholipids (e.g., aminophospholipids such as phosphatidylserineand phosphatidylethanolamine, choline phospholipids such asphosphatidylcholine and sphingomyelin, and bile acids) across a cellularmembrane by the 67118 or 67067 protein in which it is contained; and/orto regulate the activity (as defined herein) of the 67118 or 67067protein in which it is contained. Accordingly, identifying the presenceof an “E1-E2 ATPases phosphorylation site” can include isolating afragment of a 67118 or 67067 molecule (e.g., a 67118 or 67067polypeptide) and assaying for the ability of the fragment to exhibit oneof the aforementioned phosphorylation site activities.

In another embodiment, a 67118 or 67067 protein of the present inventionmay also be identified based on its ability to adopt an E1 conformationor an E2 conformation. As used herein, an “E1 conformation” of a 67118or 67067 protein includes a 3-dimensional conformation of a 67118 or67067 protein which does not exhibit 67118 or 67067 activity (e.g., theability to transport phospholipids), as defined herein. An E1conformation of a 67118 or 67067 protein usually occurs when the 67118or 67067 protein is unphosphorylated. As used herein, an “E2conformation” of a 67118 or 67067 protein includes a 3-dimensionalconformation of a 67118 or 67067 protein which exhibits 67118 or 67067activity (e.g., the ability to transport phospholipids), as definedherein. An E2 conformation of a 67118 or 67067 protein usually occurswhen the 67118 or 67067 protein is phosphorylated.

In still another embodiment, a 67118 or 67067 protein of the presentinvention is identified based on the presence of “phospholipidtransporter specific” amino acid residues. As used herein, “phospholipidtransporter specific” amino acid residues are amino acid residuesspecific to the class of phospholipid transporting P-type ATPases (asdefined in Tang, X. et al. (1996) Science 272:1495-1497). Phospholipidtransporter specific amino acid residues are not found in those P-typeATPases which transport molecules which are not phospholipids (e.g.,cations). For example, phospholipid transporter specific amino acidresidues are found at the first, second, and fifth positions of theP-type ATPase sequence 1 motif. In phospholipid transporting P-typeATPases, the first position of the P-type ATPase sequence 1 motif ispreferably E (glutamic acid), the second position is preferably T(threonine), and the fifth position is preferably L (leucine). Aphospholipid transporter specific amino acid residue is further found atthe second position of the P-type ATPase sequence 2 motif. Inphospholipid transporting P-type ATPases, the second position of theP-type ATPase sequence 2 motif is preferably F (phenylalanine).Phospholipid transporter specific amino acid residues are still furtherfound at the first, tenth, and eleventh positions of the P-type ATPasesequence 3 motif. In phospholipid transporting P-type ATPases, the firstposition of the P-type ATPase sequence 3 motif is preferably I(isoleucine), the tenth position is preferably M (methionine), and theeleventh position is preferably I (isoleucine). Phospholipid transporterspecific amino acid residues were identified in the amino acid sequenceof human 67118 (SEQ ID NO:34) at about residues 179 and 183 (within theP-type ATPase sequence 1 motif; see FIGS. 21A-B), at about residue 442(within the P-type ATPase sequence 2 motif; see FIGS. 21A-B), and atabout residues 823, 832 and 833 (within the P-type ATPase sequence 3motif; see FIGS. 21A-B). Phospholipid transporter specific amino acidresidues were identified in the amino acid sequence of human 67067 (SEQID NO:37) at about residues 175, 176, and 179 (within the P-type ATPasesequence 1 motif; see FIGS. 23A-B), at about residue 432 (within theP-type ATPase sequence 2 motif; see FIGS. 23A-B), and at about residues1180, 1189, and 1190 (within the P-type ATPase sequence 3 motif; seeFIGS. 23A-B).

Isolated polypeptides of the present invention, preferably 67118 and/or67067 polypeptides, have an amino acid sequence sufficiently identicalto the amino acid sequence of SEQ ID NO:34 or 37 or are encoded by anucleotide sequence sufficiently identical to SEQ ID NO:33, 35, 36, or38. As used herein, the term “sufficiently identical” refers to a firstamino acid or nucleotide sequence which contains a sufficient or minimumnumber of identical or equivalent (e.g., an amino acid residue which hasa similar side chain) amino acid residues or nucleotides to a secondamino acid or nucleotide sequence such that the first and second aminoacid or nucleotide sequences share common structural domains or motifsand/or a common functional activity. For example, amino acid ornucleotide sequences which share common structural domains having atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%,98%, 99% or more homology or identity across the amino acid sequences ofthe domains and contain at least one and preferably two structuraldomains or motifs, are defined herein as sufficiently identical.Furthermore, amino acid or nucleotide sequences which share at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%,99% or more homology or identity and share a common functional activityare defined herein as sufficiently identical.

In a preferred embodiment, a 67118 or 67067 protein includes at leastone or more of the following domains, sites, or motifs: a transmembranedomain, an N-terminal large extramembrane domain, a C-terminal largeextramembrane domain, an E1-E2 ATPases phosphorylation site, a P-typeATPase sequence 1 motif, a P-type ATPase sequence 2 motif, a P-typeATPase sequence 3 motif, and/or one or more phospholipid transporterspecific amino acid resides, and has an amino acid sequence at leastabout 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%,98%, 99% or more homologous or identical to the amino acid sequence ofSEQ ID NO:34 or 37. In yet another preferred embodiment, a 67118 or67067 protein includes at least one or more of the following domains,sites, or motifs: a transmembrane domain, an N-terminal largeextramembrane domain, a C-terminal large extramembrane domain, an E1-E2ATPases phosphorylation site, a P-type ATPase sequence 1 motif, a P-typeATPase sequence 2 motif, a P-type ATPase sequence 3 motif, and/or one ormore phospholipid transporter specific amino acid resides, and isencoded by a nucleic acid molecule having a nucleotide sequence whichhybridizes under stringent hybridization conditions to a complement of anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, 3, 4, or 6. In another preferred embodiment, a 67118 or 67067 proteinincludes at least one or more of the following domains, sites, ormotifs: a transmembrane domain, an N-terminal large extramembranedomain, a C-terminal large extramembrane domain, an E1-E2 ATPasesphosphorylation site, a P-type ATPase sequence 1 motif, a P-type ATPasesequence 2 motif, a P-type ATPase sequence 3 motif, and/or one or morephospholipid transporter specific amino acid resides, and has a 67118 or67067 activity.

As used interchangeably herein, a “phospholipid transporter activity” ora “67118 or 67067 activity” includes an activity exerted or mediated bya 67118 or 67067 protein, polypeptide or nucleic acid molecule on a67118 or 67067 responsive cell or on a 67118 or 67067 substrate, asdetermined in vivo or in vitro, according to standard techniques. In oneembodiment, a phospholipid transporter activity is a direct activity,such as an association with a 67118 or 67067 target molecule. As usedherein, a “target molecule” or “binding partner” is a molecule withwhich a 67118 or 67067 protein binds or interacts in nature, such that67118 or 67067-mediated function is achieved. In an exemplaryembodiment, a 67118 or 67067 target molecule is a 67118 or 67067substrate (e.g., a phospholipid, ATP, or a non-67118 or 67067 protein).A phospholipid transporter activity can also be an indirect activity,such as a cellular signaling activity mediated by interaction of the67118 or 67067 protein with a 67118 or 67067 substrate.

In a preferred embodiment, a phospholipid transporter activity is atleast one of the following activities: (i) interaction with a 67118 or67067 substrate or target molecule (e.g., a phospholipid, ATP, or anon-67118 or non-67067 protein); (ii) transport of a 67118 or 67067substrate or target molecule (e.g., an aminophospholipid such asphosphatidylserine or phosphatidylethanolamine) from one side of acellular membrane to the other; (iii) the ability to be phosphorylatedor dephosphorylated; (iv) adoption of an E1 conformation or an E2conformation; (v) conversion of a 67118 or 67067 substrate or targetmolecule to a product (e.g., hydrolysis of ATP); (vi) interaction with asecond non-67118 or non-67067 protein; (vii) modulation of substrate ortarget molecule location (e.g., modulation of phospholipid locationwithin a cell and/or location with respect to a cellular membrane);(viii) maintenance of aminophospholipid gradients; (ix) modulation ofintra- or intercellular signaling and/or gene transcription (e.g.,either directly or indirectly); and/or (x) modulation of cellularproliferation, growth, differentiation, apoptosis, absorption, orsecretion.

The nucleotide sequence of the isolated human 67118 and 67067 cDNA andthe predicted amino acid sequence of the human 67118 and 67067polypeptides are shown in SEQ ID NOs:33, 34 and 36, 37, respectively.

The human 67118 gene, which is approximately 7745 nucleotides in length,encodes a polypeptide which is approximately 1134 amino acid residues inlength. The human 67067 gene, which is approximately 7205 nucleotides inlength, encodes a polypeptide which is approximately 1588 amino acidresidues in length.

62092 family members likewise share structural and functionalcharacteristics and can be identified by said characteristics, asfollows. In another embodiment, a 62092 protein of the present inventionis identified based on the presence of a signal peptide. The predictionof such a signal peptide can be made, for example, by using the computeralgorithm SignalP (Henrik et al. (1997) Protein Eng. 10: 1-6). As usedherein, a “signal sequence” or “signal peptide” includes a peptidecontaining about 15 or more amino acids which occurs at the N-terminusof secretory and/or membrane bound proteins and which contains a largenumber of hydrophobic amino acid residues. For example, a signalsequence contains at least about 10-30 amino acid residues, preferablyabout 15-25 amino acid residues, more preferably about 18-20 amino acidresidues, and more preferably about 19 amino acid residues, and has atleast about 35-65%, preferably about 38-50%, and more preferably about40-45% hydrophobic amino acid residues (e.g., Valine, Leucine,Isoleucine or Phenylalanine). Such a “signal sequence”, also referred toin the art as a “signal peptide”, serves to direct a protein containingsuch a sequence to a lipid bilayer, and is cleaved in secreted andmembrane bound proteins. A possible signal sequence was identified inthe amino acid sequence of human 62092 at about amino acids 1-19 of SEQID NO:40.

In still another embodiment, members of the 62092 family of proteinsinclude at least one “HIT family domain” in the protein or correspondingnucleic acid molecule. As used interchangeably herein, the term “HITfamily domain” includes a protein domain having at least about 30-170amino acid residues and a bit score of at least 60.0 when comparedagainst a HIT family domain Hidden Markov Model (HMM), e.g., AccessionNumber PF01230. Preferably, a HIT family domain includes a proteindomain having an amino acid sequence of about 50-150, 70-130, 90-110, ormore preferably about 102 amino acid residues, and a bit score of atleast 80, 100, 120, 140, 160, or more preferably, 180.3. To identify thepresence of a HIT family domain in a 62092 protein, and make thedetermination that a protein of interest has a particular profile, theamino acid sequence of the protein is searched against a database ofknown protein motifs and/or domains (e.g., the HMM database). The HITfamily domain (HMM) has been assigned the PFAM Accession number PF01230.A search was performed against the HMM database resulting in theidentification of a HIT family domain in the amino acid sequence ofhuman 62092 at about residues 54-155 of SEQ ID NO:40.

A description of the Pfam database can be found in Sonhammer et al.(1997) Proteins 28:405-420, and a detailed description of HMMs can befound, for example, in Gribskov et al. (1990) Meth. Enzymol.183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; andStultz et al. (1993) Protein Sci. 2:305-314, the contents of which areincorporated herein by reference.

Preferably a HIT family domain is at least about 80-120 amino acidresidues and comprises core amino acid residues sufficient to carry outa 62092 activity, as described herein. In a preferred embodiment, a “HITfamily domain” includes at least about 90-110 amino acid residues, forexample, about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, or 110 amino acid residues,preferably, about 102 residues, and is capable of carrying out a 62092biological activity. Accordingly, identifying the presence of a “HITfamily domain” can include isolating a fragment of a 62092 molecule(e.g., a 62092 polypeptide) and assaying for the ability of the fragmentto exhibit one of the aforementioned HIT family domain activities.

In another embodiment, a 62092 protein of the present invention isidentified based on the presence of an “HIT family signature motif” inthe protein or corresponding nucleic acid molecule. The consensus for aHIT family signature motif is a protein motif and has the consensussequence[NGA]-X(4)-[GSAV]-X-[QF]-X-[LIVM]-X-H-[LIVMFYST]-H-[LIVMFT]-H-[LIVMF](2)-[PSGA](SEQ ID NO:50). The HIT family signature motif functions in nucleotidebinding and has been assigned Prosite™ Accession Number PS00892. Toidentify the presence of an HIT family signature motif in a 62092protein, and to make the determination that a protein of interest has aparticular profile, the amino acid sequence of the protein may besearched against a database of known protein domains or motifs (e.g.,the Prosite™ database) using the default parameters (available at theProSite internet website). A search was performed against the ProSitedatabase resulting in the identification of a HIT family signature motifin the amino acid sequence of human 62092 (SEQ ID NO:40) at aboutresidues 136-151.

Isolated proteins of the present invention, preferably 62092 proteins,have an amino acid sequence sufficiently homologous to the amino acidsequence of SEQ ID NO:40, or are encoded by a nucleotide sequencesufficiently homologous to SEQ ID NO:39 or 41. As used herein, the term“sufficiently homologous” refers to a first amino acid or nucleotidesequence which contains a sufficient or minimum number of identical orequivalent (e.g., an amino acid residue which has a similar side chain)amino acid residues or nucleotides to a second amino acid or nucleotidesequence such that the first and second amino acid or nucleotidesequences share common structural domains or motifs and/or a commonfunctional activity. For example, amino acid or nucleotide sequenceswhich share common structural domains having at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or morehomology or identity across the amino acid sequences of the domains andcontain at least one and preferably two structural domains or motifs,are defined herein as sufficiently homologous. Furthermore, amino acidor nucleotide sequences which share at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology oridentity and share a common functional activity are defined herein assufficiently homologous.

In a preferred embodiment, a 62092 protein includes at least one or moreof the following domains or motifs: a signal peptide, a HIT familydomain, and/or a HIT family signature motif, and has an amino acidsequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%,90%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to theamino acid sequence of SEQ ID NO:40. In yet another preferredembodiment, a 62092 protein includes at least one or more of thefollowing domains or motifs: a signal peptide, a HIT family domain,and/or a HIT family signature motif, and is encoded by a nucleic acidmolecule having a nucleotide sequence which hybridizes under stringenthybridization conditions to a complement of a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:39 or 41. In anotherpreferred embodiment, a 62092 protein includes at least one or more ofthe following domains or motifs: a signal peptide, a HIT family domain,and/or a HIT family signature motif, and has a 62092 activity.

As used interchangeably herein, a “62092 activity”, “biological activityof 62092” or “functional activity of 62092”, includes an activityexerted or mediated by a 62092 protein, polypeptide or nucleic acidmolecule on a 62092 responsive cell or on a 62092 substrate, asdetermined in vivo or in vitro, according to standard techniques. In oneembodiment, a 62092 activity is a direct activity, such as anassociation with a 62092 target molecule. As used herein, a “targetmolecule” or “binding partner” is a molecule with which a 62092 proteinbinds or interacts in nature, such that 62092-mediated function isachieved. In an exemplary embodiment, a 62092 target molecule is a 62092substrate (e.g., a nucleotide such as a purine mononucleotide (e.g.,adenosine, AMP, GMP, or 8Br-AMP) or an dinucleoside polyphosphate (e.g.,ApppA, AppppA, or AppppG)). A 62092 activity can also be an indirectactivity, such as a cellular signaling activity mediated by interactionof the 62092 protein with a 62092 substrate. For example, a 62092protein:substrate complex can interact with a downstream signalingmolecule or target in order to indirectly effect a 62092 biologicalactivity.

In a preferred embodiment, a 62092 activity is at least one of thefollowing activities: (i) interaction with a 62092 substrate or targetmolecule (e.g., a nucleotide such as a purine mononucleotide or anucleoside polyphosphate), or a non-62092 protein); (ii) conversion of a62092 substrate or target molecule to a product (e.g., cleavage of adinucleoside polyphosphate); (iii) interaction with a second non-62092protein; (iv) sensation of cellular stress signals; (v) regulation ofsubstrate or target molecule availability or activity; (vi) modulationof intra- or intercellular signaling and/or gene transcription (e.g.,either directly or indirectly); and/or (vii) modulation of cellularproliferation, growth, differentiation, and/or apoptosis.

The nucleotide sequence of the isolated human 62092 cDNA and thepredicted amino acid sequence encoded by the 62092 cDNA are shown in SEQID NOs:39 and 40, respectively.

The human 62092 gene, which is approximately 978 nucleotides in length,encodes a protein having a molecular weight of approximately 6.9 kD andwhich is approximately 163 amino acid residues in length.

Various aspects of the invention are described in further detail inlater subsections.

Chapter VIII.FBH58295FL, A Novel Human Amino Acid Transporter and UsesThereof

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel amino acid transporter family members, referred to herein as“Human Amino Acid Transporter” or “HAAT” nucleic acid and proteinmolecules. The HAAT nucleic acid and protein molecules of the presentinvention are useful as modulating agents in regulating a variety ofcellular processes, e.g., protein synthesis, hormone metabolism, nervetransmission, cellular activation, regulation of cell growth, productionof metabolic energy, synthesis of purines and pyrimidines, nitrogenmetabolism, and/or biosynthesis of urea. Accordingly, in one aspect,this invention provides isolated nucleic acid molecules encoding HAATproteins or biologically active portions thereof, as well as nucleicacid fragments suitable as primers or hybridization probes for thedetection of HAAT-encoding nucleic acids.

In one embodiment, the invention features an isolated nucleic acidmolecule that includes the nucleotide sequence set forth in SEQ ID NO:51or 53. In another embodiment, the invention features an isolated nucleicacid molecule that encodes a polypeptide including the amino acidsequence set forth in SEQ ID NO:52.

In still other embodiments, the invention features isolated nucleic acidmolecules including nucleotide sequences that are substantiallyidentical (e.g., 80% identical) to the nucleotide sequence set forth asSEQ ID NO:51 or 53. The invention further features isolated nucleic acidmolecules including at least 30 contiguous nucleotides of the nucleotidesequence set forth as SEQ ID NO: 51 or 53. In another embodiment, theinvention features isolated nucleic acid molecules which encode apolypeptide including an amino acid sequence that is substantiallyidentical (e.g., 80% identical) to the amino acid sequence set forth asSEQ ID NO:52. Also featured are nucleic acid molecules which encodeallelic variants of the polypeptide having the amino acid sequence setforth as SEQ ID NO:52. In addition to isolated nucleic acid moleculesencoding full-length polypeptides, the present invention also featuresnucleic acid molecules which encode fragments, for example, biologicallyactive or antigenic fragments, of the full-length polypeptides of thepresent invention (e.g., fragments including at least 10 contiguousamino acid residues of the amino acid sequence of SEQ ID NO:52). Instill other embodiments, the invention features nucleic acid moleculesthat are complementary to, antisense to, or hybridize under stringentconditions to the isolated nucleic acid molecules described herein.

In a related aspect, the invention provides vectors including theisolated nucleic acid molecules described herein (e.g., HAAT-encodingnucleic acid molecules). Such vectors can optionally include nucleotidesequences encoding heterologous polypeptides. Also featured are hostcells including such vectors (e.g., host cells including vectorssuitable for producing HAAT nucleic acid molecules and polypeptides).

In another aspect, the invention features isolated HAAT polypeptidesand/or biologically active or antigenic fragments thereof. Exemplaryembodiments feature a polypeptide including the amino acid sequence setforth as SEQ ID NO:52, a polypeptide including an amino acid sequence atleast 80% identical to the amino acid sequence set forth as SEQ IDNO:52, a polypeptide encoded by a nucleic acid molecule including anucleotide sequence at least 80% identical to the nucleotide sequenceset forth as SEQ ID NO:51 or 53. Also featured are fragments of thefull-length polypeptides described herein (e.g., fragments including atleast 10 contiguous amino acid residues of the sequence set forth as SEQID NO:52) as well as allelic variants of the polypeptide having theamino acid sequence set forth as SEQ ID NO:52.

The HAAT polypeptides and/or biologically active or antigenic fragmentsthereof, are useful, for example, as reagents or targets in assaysapplicable to treatment and/or diagnosis of HAAT associated or relateddisorders. In one embodiment, a HAAT polypeptide or fragment thereof hasa HAAT activity. In another embodiment, a HAAT polypeptide or fragmentthereof has at least one or more of the following domains, sites, ormotifs: a transmembrane domain, a transmembrane amino acid transporterdomain, and optionally, has a HAAT activity. In a related aspect, theinvention features antibodies (e.g., antibodies which specifically bindto any one of the polypeptides, as described herein) as well as fusionpolypeptides including all or a fragment of a polypeptide describedherein.

The present invention further features methods for detecting HAATpolypeptides and/or HAAT nucleic acid molecules, such methods featuring,for example, a probe, primer or antibody described herein. Also featuredare kits for the detection of HAAT polypeptides and/or HAAT nucleic acidmolecules. In a related aspect, the invention features methods foridentifying compounds which bind to and/or modulate the activity of aHAAT polypeptide or HAAT nucleic acid molecule described herein. Alsofeatured are methods for modulating a HAAT activity.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel amino acid transporter family members, referred to herein as“Human Amino Acid Transporter” or “HAAT” nucleic acid and proteinmolecules, also referred to interchangeably herein as “FBH5829FL”nucleic acid and protein molecules. These novel molecules are capable oftransporting alanine, serine, proline, glutamine, and N-methyl aminoacids across cellular membranes and, thus, play a role in or function ina variety of cellular processes, e.g., protein synthesis, hormonemetabolism, nerve transmission, cellular activation, regulation of cellgrowth, production of metabolic energy, synthesis of purines andpyrimidines, nitrogen metabolism, and/or biosynthesis of urea. Thus, theHAAT molecules of the present invention provide novel diagnostic targetsand therapeutic agents to control HAAT-associated disorders, as definedherein.

The term “treatment” as used herein, is defined as the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease or the predisposition toward disease. Atherapeutic agent includes, but is not limited to, small molecules,peptides, antibodies, ribozymes and antisense oligonucleotides.

The term “family” when referring to the protein and nucleic acidmolecules of the invention is intended to mean two or more proteins ornucleic acid molecules having a common structural domain or motif andhaving sufficient amino acid or nucleotide sequence homology as definedherein. Such family members can be naturally or non-naturally occurringand can be from either the same or different species. For example, afamily can contain a first protein of human origin as well as otherdistinct proteins of human origin or alternatively, can containhomologues of non-human origin, e.g., rat or mouse proteins. Members ofa family can also have common functional characteristics.

For example, the family of HAAT polypeptides comprise at least one“transmembrane domain” and preferably at least two, three, four, five,fix, seven, eight, nine, ten, or eleven transmembrane domains. As usedherein, the term “transmembrane domain” includes an amino acid sequenceof about 15-45 amino acid residues in length which spans the plasmamembrane. More preferably, a transmembrane domain includes about atleast 15, 20, 25, 30, 35, 40, or 45 amino acid residues and spans theplasma membrane. Transmembrane domains are rich in hydrophobic residues,and typically have an alpha-helical structure. In a preferredembodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the aminoacids of a transmembrane domain are hydrophobic, e.g., leucines,isoleucines, alanines, valines, phenylalanines, prolines or methionines.Transmembrane domains are described in, for example, Zagotta W. N. etal, (1996) Annual Rev. Neurosci. 19: 235-263, the contents of which areincorporated herein by reference. A MEMSAT analysis and a structural,hydrophobicity, and antigenicity analysis resulted in the identificationof ten transmembrane domains in the amino acid sequence of HAAT (SEQ IDNO:52) at about residues 68-92, 135-156, 190-207, 214-232, 256-274,287-308, 334-356, 373-390, 397-421, and 435-453 as set forth in FIGS. 26and 28. Manual analysis of the amino acid sequence of human HAATresulted in the identification of an additional transmembrane domain atamino acids 42-65 of SEQ ID NO:52.

The family of HAAT polypeptides also comprises at least one“transmembrane amino acid transporter protein domain.” As used herein,the term “transmembrane amino acid transporter protein domain” includestransmembrane domains found in amino acid sequences that are involved inthe transport of amino acids across a membrane. There are a wide rangeof amino acid transporter proteins that may be classified into amultitude of different amino acid transporter systems. A listing of someof the different amino acid transporter systems follows.

System A

System A transports small aliphatic amino acids including alanine,serine, proline, glutamine and is wide expressed in mammalian cellsincluding myocytes and hepatocytes. In the intestine, system A islocalized to basolateral membranes where it absorbs amino acids from theblood for the metabolic requirement of enterocytes. (Stevens, et al.(1984) A. Rev. Physiol. 46:417-433). System A is Na⁺-coupled, toleratesLi⁺ and is pH sensitive. (Christensen, et al. (1965) J. Biol. Chem.240:3609-3616). System A recognize N-methyl amino acids, and(N-methylamino)-α-isobutyric acid (MeAIB) is a characteristic substrate.System A is regulated by amino acid deprivation, hormones, growthfactors and hyperosmotic stress. For example, insulin stimulates systemA activity in both liver and skeletal muscle, and glucagon alsostimulates it synergistically in hepatocytes. (Le Cam, et al. (1978)Diabetologia 15:1835-1853).

System ASC

System ASC provides cell with the amino acids alanine, threonine,serine, cysteine. System ASC is distinguishable from system A because(1) it does not recognize (N-methylamino)-α-isobutyric acid (MeAIB), and(2) neutral amino acid uptake is relatively pH-insensitive.

Systems B, B⁰, and B⁰⁺

Systems B, B⁰, and B⁰⁺ mediate the absorption of aliphate,branched-chain and aromatic amino acids. B⁰⁺ also accepts dibasic aminoacids. (Van Winkle, et al. (1988) Biochim. Biophys. Acta 947:173-208.)Systems B, B⁰, and B⁰⁺ are Na⁺-dependent. Systems B and B⁰ have abroader specificity for neutral amino acids than systems A and ASC.Systems B and B⁰ are present in intestinal and renal epithelialbrush-border membranes. (Stevens, et al. (1984) A. Rev. Physiol.46:417-433). System B⁰⁺ is both Na⁺ and Cl⁻-coupled. (Van Winkle (1985)J. Biol. Chem. 260:12118-12123.)

System b⁰⁺

The mouse blastocyst transport system b⁰⁺ mediates Na⁺ independent, highaffinity transport of neutral and dibasic amino acids. It is expressedin kidney and intestinal epithelia.

System N

System N is Na⁺ coupled and specific for neutral amino acids. It has amore restricted tissue distribution than systems A, ASC, B, B⁰, and B⁰⁺.It is expressed in liver and muscle. In liver, system N is involved inthe transport of glutamine, asparagine and histidine and it plays animportant role in glutamine metabolism. Kilberg, et al. (1980) J. Biol.Chem. 255:4011-4019.

System GLY

System GLY is specific for glycine and sarcosine and is found in liver,erythrocytes, and brain.

System β

System β is specific for β-amino acids and taurine. Given its highabundance in the brain, it is thought to play a role inneurotransmission.

The Imino System

The iminio system is specific for proline and was described in brushborder membranes of intestinal enterocytes. The iminio system accountsfor 60% of the Na⁺-dependent uptake of proline in brush-border membranesand is specific for imino acids and MeAIB.

System L

System L transport branched-chain and aromatic amino acids. System L isNa⁺-independent. In the brain, system L is the major transport system ofthe blood-brain barrier and of glial cells. The bicyclic amino acid2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) is a characteristicsubstrate of system L.

System X⁻ _(AG)

System X⁻ _(AG) is an electrogenic Na⁺-dependent acidic amino acidtransport system that has been found in both epithelial cells andneurons. In the central nervous system, glutamate plays an importantrole as excitatory neurotransmitter. To terminate signal transmission,glutamate is removed from the extracellular fluid in the synaptic cleftsurrounding the receptors by specialized uptake systems in neurons andglial cells because there are no enzymatic pathways for transmitterinactivation.

System y⁺

System y⁺ takes up cationic acid. System y⁺ also takes up some neutralamino acids in the presence of Na⁺, resulting in electrogenic transport.

System x⁻ _(c)

System x⁻ _(c) is a Na⁺-independent, Cl⁻ dependent, cystine/glutamateexchange. System x⁻ _(c) has been found in fibroblasts, macrophages,endothelial cells, glial cells, and hepatocytes.

Isolated proteins of the present invention, preferably HAAT proteins,have an amino acid sequence sufficiently homologous to the amino acidsequence of SEQ ID NO:52, or are encoded by a nucleotide sequencesufficiently homologous to SEQ ID NO:51 or 53. As used herein, the term“sufficiently homologous” refers to a first amino acid or nucleotidesequence which contains a sufficient or minimum number of identical orequivalent (e.g., an amino acid residue which has a similar side chain)amino acid residues or nucleotides to a second amino acid or nucleotidesequence such that the first and second amino acid or nucleotidesequences share common structural domains or motifs and/or a commonfunctional activity. For example, amino acid or nucleotide sequenceswhich share common structural domains having at least 75%, 80%, 85%,85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology or identity acrossthe amino acid sequences of the domains and contain at least one andpreferably two structural domains or motifs, are defined herein assufficiently homologous. Furthermore, amino acid or nucleotide sequenceswhich share at least 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% ormore homology or identity and share a common functional activity aredefined herein as sufficiently homologous. In a preferred embodiment,amino acid or nucleotide sequences share percent identity across thefull or entire length of the amino acid or nucleotide sequence beingaligned, for example, when the sequences are globally aligned (e.g., asdetermined by the ALIGN algorithm as defined herein).

In a preferred embodiment, a HAAT protein includes at least one or moreof the following domains, sites, or motifs: a transmembrane domain, atransmembrane amino acid transporter domain and has an amino acidsequence at least about 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or more homologous or identical to the amino acid sequence of SEQ IDNO:52.

As used interchangeably herein, a “HAAT activity”, “amino acidtransporter activity”, “biological activity of HAAT”, or “functionalactivity of HAAT”, includes an activity exerted or mediated by a HAATprotein, polypeptide or nucleic acid molecule on a HAAT responsive cellor on a HAAT substrate, as determined in vivo or in vitro, according tostandard techniques. In one embodiment, a HAAT activity is a directactivity, such as an association with a HAAT target molecule. As usedherein, a “target molecule” or “binding partner” is a molecule withwhich a HAAT protein binds or interacts in nature, such thatHAAT-mediated function is achieved. A HAAT target molecule can be anon-HAAT molecule or a HAAT protein or polypeptide of the presentinvention. In an exemplary embodiment, a HAAT target molecule is a HAATsubstrate (e.g., an amino acid). A HAAT activity can also be an indirectactivity, such as a protein synthesis activity mediated by interactionof the HAAT protein with a HAAT substrate.

In a preferred embodiment, a HAAT activity is at least one of thefollowing activities: (i) interaction with a HAAT substrate or targetmolecule (e.g., an amino acid); (ii) transport of a HAAT substrate ortarget molecule (e.g., an amino acid) from one side of a cellularmembrane to the other; (iii) conversion of a HAAT substrate or targetmolecule to a product (e.g., glucose production); (iv) interaction witha second non-HAAT protein; (v) modulation of substrate or targetmolecule location (e.g., modulation of amino acid location within a celland/or location with respect to a cellular membrane); (vi) maintenanceof amino acid gradients; (vii) modulation of hormone metabolism and/ornerve transmission (e.g., either directly or indirectly); (viii)modulation of cellular proliferation, growth, differentiation, andproduction of metabolic energy; and/or (ix) modulation of amino acidhomeostasis.

The nucleotide sequence of the isolated human HAAT cDNA and thepredicted amino acid sequence encoded by the HAAT cDNA are shown in SEQID NO:51 and 52, respectively.

The human HAAT gene, which is approximately 2397 nucleotides in length,encodes a protein which is approximately 485 amino acid residues inlength.

Various aspects of the invention are described in further detail inlater subsections.

Chapter IX. 57255 and 57255alt, Novel Human Sugar Transporters and UsesTherefor

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel human sugar transporter family members, referred to herein as“human sugar transporters,” e.g., “human sugar transporter-4” and “humansugar transporter-5” or “HST-4” and “HST-5,” nucleic acid andpolypeptide molecules. The HST-4 and HST-5 nucleic acid and polypeptidemolecules of the present invention are useful as modulating agents inregulating a variety of cellular processes, e.g., sugar homeostasis.Accordingly, in one aspect, this invention provides isolated nucleicacid molecules encoding HST-4 and HST-5 polypeptides or biologicallyactive portions thereof, as well as nucleic acid fragments suitable asprimers or hybridization probes for the detection of HST-4- andHST-5-encoding nucleic acids.

In one embodiment, the invention features an isolated nucleic acidmolecule that includes the nucleotide sequence set forth in SEQ IDNO:54, 56, 57, or 59. In another embodiment, the invention features anisolated nucleic acid molecule that encodes a polypeptide including theamino acid sequence set forth in SEQ ID NO:55 or 58.

In still other embodiments, the invention features isolated nucleic acidmolecules including nucleotide sequences that are substantiallyidentical (e.g., 60% identical) to the nucleotide sequence set forth asSEQ ID NO: 54, 56, 57, or 59. The invention further features isolatednucleic acid molecules including at least 50 contiguous nucleotides ofthe nucleotide sequence set forth as SEQ ID NO: 54, 56, 57, or 59. Inanother embodiment, the invention features isolated nucleic acidmolecules which encode a polypeptide including an amino acid sequencethat is substantially identical (e.g., 60% identical) to the amino acidsequence set forth as SEQ ID NO:55 or 58. The present invention alsofeatures nucleic acid molecules which encode allelic variants of thepolypeptide having the amino acid sequence set forth as SEQ ID NO:55 or58. In addition to isolated nucleic acid molecules encoding full-lengthpolypeptides, the present invention also features nucleic acid moleculeswhich encode fragments, for example, biologically active or antigenicfragments, of the full-length polypeptides of the present invention(e.g., fragments including at least 10 contiguous amino acid residues ofthe amino acid sequence of SEQ ID NO:55 or 58). In still otherembodiments, the invention features nucleic acid molecules that arecomplementary to, antisense to, or hybridize under stringent conditionsto the isolated nucleic acid molecules described herein.

In another aspect, the invention provides vectors including the isolatednucleic acid molecules described herein (e.g., HST-4- and HST-5-encodingnucleic acid molecules). Such vectors can optionally include nucleotidesequences encoding heterologous polypeptides. Also featured are hostcells including such vectors (e.g., host cells including vectorssuitable for producing HST-4 and HST-5 nucleic acid molecules andpolypeptides).

In another aspect, the invention features isolated HST-4 and HST-5polypeptides and/or biologically active or antigenic fragments thereof.Exemplary embodiments feature a polypeptide including the amino acidsequence set forth as SEQ ID NO:55 or 58, a polypeptide including anamino acid sequence at least 60% identical to the amino acid sequenceset forth as SEQ ID NO:55 or 58, a polypeptide encoded by a nucleic acidmolecule including a nucleotide sequence at least 60% identical to thenucleotide sequence set forth as SEQ ID NO: 54, 56, 57, or 59. Alsofeatured are fragments of the full-length polypeptides described herein(e.g., fragments including at least 10 contiguous amino acid residues ofthe sequence set forth as SEQ ID NO:55 or 58) as well as allelicvariants of the polypeptide having the amino acid sequence set forth asSEQ ID NO:55 or 58.

The HST-4 and HST-5 polypeptides and/or biologically active or antigenicfragments thereof, are useful, for example, as reagents or targets inassays applicable to treatment and/or diagnosis of HST-4 and HST-5mediated or related disorders. In one embodiment, HST-4 and/or HST-5polypeptides or fragments thereof, have an HST-4 and/or HST-5 activity.In another embodiment, HST-4 and/or HST-5 polypeptides or fragmentsthereof, have at least one, preferably two, three, four, five, six,seven, eight, nine, ten, or eleven transmembrane domains and/or a sugartransporter family domain, and optionally, have an HST-4 and/or HST-5activity. In a related aspect, the invention features antibodies (e.g.,antibodies which specifically bind to any one of the polypeptidesdescribed herein) as well as fusion polypeptides including all or afragment of a polypeptide described herein.

The present invention further features methods for detecting HST-4and/or HST-5 polypeptides and/or HST-4 and/or HST-5 nucleic acidmolecules, such methods featuring, for example, a probe, primer orantibody described herein. Also featured are kits e.g., kits for thedetection of HST-4 and/or HST-5 polypeptides and/or HST-4 and/or HST-5nucleic acid molecules. In a related aspect, the invention featuresmethods for identifying compounds which bind to and/or modulate theactivity of an HST-4 and/or an HST-5 polypeptide or HST-4 and/or HST-5nucleic acid molecule described herein. Further featured are methods formodulating an HST-4 and/or an HST-5 activity.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as “human sugar transporter-4” and“human sugar transporter-5” or “HST-4” and “HST-5” nucleic acid andpolypeptide molecules, which are novel members of the sugar transporterfamily. These novel molecules are splice variants which have resultedfrom alternative splicing of the same gene. These novel molecules arecapable of, for example, modulating a transporter mediated activity(e.g., a sugar transporter mediated activity) in a cell, e.g., a livercell, fat cell, muscle cell, or blood cell, such as an erythrocyte.These novel molecules are capable of transporting molecules, e.g.,hexoses such as D-glucose, D-fructose, D-galactose or mannose acrossbiological membranes and, thus, play a role in or function in a varietyof cellular processes, e.g., maintenance of sugar homeostasis. As usedherein, a “sugar transporter” includes a protein or polypeptide which isinvolved in transporting a molecule, e.g., a monosaccharide such asD-glucose, D-fructose, D-galactose or mannose, across the plasmamembrane of a cell, e.g., a liver cell, fat cell, muscle cell, or bloodcell, such as an erythrocyte. Sugar transporters regulate sugarhomeostasis in a cell and, typically, have sugar substrate specificity.Examples of sugar transporters include glucose transporters, fructosetransporters, and galactose transporters.

As used herein, a “sugar transporter mediated activity” includes anactivity which involves a sugar transporter, e.g., a sugar transporterin a liver cell, fat cell, muscle cell, or blood cell, such as anerythrocyte. Sugar transporter mediated activities include the transportof sugars, e.g., D-glucose, D-fructose, D-galactose or mannose, into andout of cells; the stimulation of molecules that regulate glucosehomeostasis (e.g., insulin and glucagon), from cells, e.g., pancreaticcells; and the participation in signal transduction pathways associatedwith sugar metabolism.

As the HST-4 and HST-5 molecules of the present invention are sugartransporters, they may be useful for developing novel diagnostic andtherapeutic agents for sugar transporter associated disorders. As usedherein, the term “sugar transporter associated disorder” includes adisorder, disease, or condition which is characterized by an aberrant,e.g., upregulated or downregulated, sugar transporter mediated activity.Sugar transporter associated disorders typically result in, e.g.,upregulated or downregulated, sugar levels in a cell. Examples of sugartransporter associated disorders include disorders associated with sugarhomeostasis, such as obesity, anorexia, type-1 diabetes, type-2diabetes, hypoglycemia, glycogen storage disease (Von Gierke disease),type I glycogenosis, bipolar disorder, seasonal affective disorder, andcluster B personality disorders.

The term “family” when referring to the polypeptide and nucleic acidmolecules of the invention is intended to mean two or more polypeptidesor nucleic acid molecules having a common structural domain or motif andhaving sufficient amino acid or nucleotide sequence homology as definedherein. Such family members can be naturally or non-naturally occurringand can be from either the same or different species. For example, afamily can contain a first polypeptide of human origin, as well asother, distinct polypeptides of human origin or alternatively, cancontain homologues of non-human origin, e.g., mouse or monkeypolypeptides. Members of a family may also have common functionalcharacteristics.

For example, the family of HST-4 and HST-5 polypeptides comprise atleast one “transmembrane domain” and at least one, preferably two,three, four, five, six, seven, eight, nine, ten, or eleven transmembranedomains. As used herein, the term “transmembrane domain” includes anamino acid sequence of about 20-45 amino acid residues in length whichspans the plasma membrane. More preferably, a transmembrane domainincludes about at least 20, 25, 30, 35, 40, or 45 amino acid residuesand spans the plasma membrane. Transmembrane domains are rich inhydrophobic residues, and typically have an alpha-helical structure. Ina preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more ofthe amino acids of a transmembrane domain are hydrophobic, e.g.,leucines, isoleucines, alanines, valines, phenylalanines, prolines ormethionines. Transmembrane domains are described in, for example,Zagotta W. N. et al, (1996) Annual Rev. Neurosci. 19: 235-263, thecontents of which are incorporated herein by reference. A MEMSAT andadditional analyses resulted in the identification of ten transmembranedomains in the amino acid sequence of human HST-4 (SEQ ID NO:55) atabout residues 25-49, 62-80, 92-113, 126-143, 154-178, 186-202, 278-298,318-337, 372-395, and 402-423. A MEMSAT and additional analyses resultedin the identification of eleven transmembrane domains in the amino acidsequence of human HST-5 (SEQ ID NO:58) at about residues 30-51, 62-84,92-111, 126-143, 154-178, 186-202, 240-260, 276-296, 316-335, 370-393,and 400-421.

Accordingly, HST-4 and HST-5 polypeptides having at least 50-60%homology, preferably about 60-70%, more preferably about 70-80%, orabout 80-90% homology with at least one, preferably at least two, three,four, five, six, seven, eight, nine, ten, or eleven transmembranedomains of human HST-4 and HST-5, respectively are within the scope ofthe invention.

In another embodiment, an HST-4 and/or HST-5 molecule of the presentinvention is identified based on the presence of at least one “sugartransporter family domain.” As used herein, the term “sugar transporterfamily domain” includes a protein domain having at least about 300-600amino acid residues and a sugar transporter mediated activity.Preferably, a sugar transporter family domain includes a polypeptidehaving an amino acid sequence of about 350-550, 400-550, or morepreferably, about 408 or 406 amino acid residues and a sugar transportermediated activity. To identify the presence of a sugar transporterfamily domain in an HST-4 and/or an HST-5 protein, and make thedetermination that a protein of interest has a particular profile, theamino acid sequence of the protein may be searched against a database ofknown protein domains (e.g., the PFAM HMM database). A PFAM sugartransporter family domain has been assigned the PFAM Accession PF00083.A search was performed against the PFAM HMM database resulting in theidentification of a sugar transporter family domain in the amino acidsequence of human HST-4 at about residues 23-431 of SEQ ID NO:55 and inthe amino acid sequence of human HST-5 at about residues 23-429 of SEQID NO:58.

Preferably a “sugar transporter family domain” has a “sugar transportermediated activity” as described herein. For example, a sugar transporterfamily domain may have the ability to bind a monosaccharide (e.g.,D-glucose, D-fructose, D-galactose and/or mannose); the ability totransport a monosaccharide (e.g., D-glucose, D-fructose, D-galactoseand/or mannose) in a constitutive manner or in response to stimuli(e.g., insulin) across a cell membrane (e.g., a liver cell membrane, fatcell membrane, muscle cell membrane, and/or blood cell membrane, such asan erythrocyte membrane); the ability to mediate trans-epithelialmovement; and/or the ability to modulate sugar homeostasis in a cell.Accordingly, identifying the presence of a “sugar transporter familydomain” can include isolating a fragment of an HST-4 and/or an HST-5molecule (e.g., an HST-4 and/or an HST-5 polypeptide) and assaying forthe ability of the fragment to exhibit one of the aforementioned sugartransporter mediated activities.

A description of the PFAM database can be found in Sonhammer et al.(1997) Proteins 28:405-420 and a detailed description of HMMs can befound, for example, in Gribskov et al. (1990) Meth. Enzymol.183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; andStultz et al. (1993) Protein Sci. 2:305-314, the contents of which areincorporated herein by reference.

In a preferred embodiment, the HST-4 and/or HST-5 molecules of theinvention include at least one, preferably two, even more preferably atleast three, four, five, six, seven, eight, nine, ten, or eleventransmembrane domain(s) and/or at least one sugar transporter familydomain.

Isolated polypeptides of the present invention, preferably HST-4 orHST-5 polypeptides, have an amino acid sequence sufficiently identicalto the amino acid sequence of SEQ ID NO:55 or 58 or are encoded by anucleotide sequence sufficiently identical to SEQ ID NO: 54, 56, 57, or59. As used herein, the term “sufficiently identical” refers to a firstamino acid or nucleotide sequence which contains a sufficient or minimumnumber of identical or equivalent (e.g., an amino acid residue which hasa similar side chain) amino acid residues or nucleotides to a secondamino acid or nucleotide sequence such that the first and second aminoacid or nucleotide sequences share common structural domains or motifsand/or a common functional activity. For example, amino acid ornucleotide sequences which share common structural domains having atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more homology or identity across theamino acid sequences of the domains and contain at least one andpreferably two structural domains or motifs, are defined herein assufficiently identical. Furthermore, amino acid or nucleotide sequenceswhich share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology or identityand share a common functional activity are defined herein assufficiently identical.

In a preferred embodiment, an HST-4 and/or HST-5 polypeptide includes atleast one or more of the following domains: a transmembrane domainand/or a sugar transporter family domain, and has an amino acid sequenceat least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identicalto the amino acid sequence of SEQ ID NO:55 or 58. In yet anotherpreferred embodiment, an HST-4 and/or an HST-5 polypeptide includes atleast one or more of the following domains: a transmembrane domainand/or a sugar transporter family domain, and is encoded by a nucleicacid molecule having a nucleotide sequence which hybridizes understringent hybridization conditions to a complement of a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO: 54, 56, 57, or59. In another preferred embodiment, an HST-4 and/or an HST-5polypeptide includes at least one or more of the following domains: atransmembrane domain and/or a sugar transporter family domain, and hasan HST-4 and/or an HST-5 activity.

As used interchangeably herein, an “HST-4 activity”, “HST-5 activity”,“biological activity of HST-4”, “biological activity of HST-5”,“functional activity of HST-4” or “functional activity of HST-5” refersto an activity exerted by an HST-4 and/or HST-5 polypeptide or nucleicacid molecule on an HST-4 and/or HST-5 responsive cell or tissue, or onan HST-4 and/or HST-5 polypeptide substrate, as determined in vivo, orin vitro, according to standard techniques. In one embodiment, an HST-4and/or HST-5 activity is a direct activity, such as an association withan HST-4- and/or HST-5-target molecule. As used herein, a “substrate,”“target molecule,” or “binding partner” is a molecule with which anHST-4 and/or HST-5 polypeptide binds or interacts in nature, such thatHST-4- and/or HST-5-mediated function is achieved. An HST-4 and/or HST-5target molecule can be a non-HST-4 and/or a non-HST-5 molecule or anHST-4 and/or HST-5 polypeptide or polypeptide of the present invention.In an exemplary embodiment, an HST-4 and/or HST-5 target molecule is anHST-4 and/or HST-5 ligand, e.g., a sugar transporter ligand suchD-glucose, D-fructose, D-galactose, and/or mannose. Alternatively, anHST-4 and/or HST-5 activity is an indirect activity, such as a cellularsignaling activity mediated by interaction of the HST-4 and/or HST-5polypeptide with an HST-4 and/or HST-5 ligand. The biological activitiesof HST-4 and/or HST-5 are described herein. For example, the HST-4and/or HST-5 polypeptides of the present invention can have one or moreof the following activities: (1) bind a monosaccharide, e.g., D-glucose,D-fructose, D-galactose, and/or mannose; (2) transport monosaccharidesacross a cell membrane; (3) influence insulin and/or glucagon secretion;(4) maintain sugar homeostasis in a cell; and (5) mediatetrans-epithelial movement in a cell. Moreover, in a preferredembodiment, HST-4 and/or HST-5 molecules of the present invention, HST-4and/or HST-5 antibodies, HST-4 and/or HST-5 modulators are useful in atleast one of the following: (1) modulation of insulin sensitivity; (2)modulation of blood sugar levels; (3) treatment of blood sugar leveldisorders (e.g., diabetes); and/or (4) modulation of insulin resistance.

The nucleotide sequence of the isolated human HST-4 and HST-5 cDNAs andthe predicted amino acid sequences of the human HST-4 and HST-5polypeptides are shown in SEQ ID NOs:54 and 55, and SEQ ID NOs:57 and58, respectively.

The human HST-4 gene, which is approximately 2565 nucleotides in length,encodes a polypeptide which is approximately 438 amino acid residues inlength. The human HST-5 gene, which is approximately 2558 nucleotides inlength, encodes a polypeptide which is approximately 436 amino acidresidues in length.

Various aspects of the invention are described in further detail in thefollowing subsections:

Chapter X. Further Embodiments of MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and HST-5

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 polypeptides or biologically activeportions thereof, as well as nucleic acid fragments sufficient for useas hybridization probes to identify MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4- and/or HST-5-encodingnucleic acid molecules (e.g., MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 mRNA) and fragmentsfor use as PCR primers for the amplification or mutation of MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 nucleic acid molecules. As used herein, the term “nucleicacid molecule” is intended to include DNA molecules (e.g., cDNA orgenomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The term “isolated nucleic acid molecule” includes nucleic acidmolecules which are separated from other nucleic acid molecules whichare present in the natural source of the nucleic acid. For example, withregards to genomic DNA, the term “isolated” includes nucleic acidmolecules which are separated from the chromosome with which the genomicDNA is naturally associated. Preferably, an “isolated” nucleic acid isfree of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7, 9,12, 14, 15, 17, 19, 21, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 51, 53,54, 56, 57, or 59, or a portion thereof, can be isolated using standardmolecular biology techniques and the sequence information providedherein. Using all or a portion of the nucleic acid sequence of SEQ IDNO:1, 3, 4, 6, 7, 9, 12, 14, 15, 17, 19, 21, 27, 29, 30, 32, 33, 35, 36,38, 39, 41, 51, 53, 54, 56, 57,or 59, as a hybridization probe, MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 nucleic acid molecules can be isolated using standardhybridization and cloning techniques (e.g., as described in Sambrook,J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A LaboratoryManual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO: 1, 3, 4, 6, 7, 9, 12, 14, 15, 17, 19, 21, 27, 29, 30, 32, 33, 35,36, 38, 39, 41, 51, 53, 54, 56, 57, or 59, can be isolated by thepolymerase chain reaction (PCR) using synthetic oligonucleotide primersdesigned based upon the sequence of SEQ ID NO:1, 3, 4, 6, 7, 9, 12, 14,15, 17, 19, 21, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 51, 53, 54, 56,57,or 59.

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5nucleotide sequences can be prepared by standard synthetic techniques,e.g., using an automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO: 1 or 3.This cDNA may comprise sequences encoding the human MTP-1 protein (i.e.,“the coding region”, from nucleotides 165-6599), as well as 5′untranslated sequences (nucleotides 1-164) and 3′ untranslated sequences(nucleotides 6600-6768) of SEQ ID NO: 1. Alternatively, the nucleic acidmolecule can comprise only the coding region of SEQ ID NO: 1 (e.g.,nucleotides 165-6599, corresponding to SEQ ID NO:3).

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NO: 1 or 3, or a portion of anyof these nucleotide sequences. A nucleic acid molecule which iscomplementary to the nucleotide sequence shown in SEQ ID NO: 1 or 3, isone which is sufficiently complementary to the nucleotide sequence shownin SEQ ID NO:1 or 3, such that it can hybridize to the nucleotidesequence shown in SEQ ID NO: 1 or 3, respectively, thereby forming astable duplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the present invention comprises a nucleotide sequence which is atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or more identical to the entire length of the nucleotidesequence shown in SEQ ID NO: 1 or 3, or a portion of any of thesenucleotide sequences.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO: 1 or 3, for example,a fragment which can be used as a probe or primer or a fragment encodinga portion of an MTP-1 protein, e.g., a biologically active portion of anMTP-1 protein. The nucleotide sequence determined from the cloning ofthe MTP-1 gene allows for the generation of probes and primers designedfor use in identifying and/or cloning other MTP-1 family members, aswell as MTP-1 homologues from other species. The probe/primer typicallycomprises substantially purified oligonucleotide. The oligonucleotidetypically comprises a region of nucleotide sequence that hybridizesunder stringent conditions to at least about 12 or 15, preferably about20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75consecutive nucleotides of a sense sequence of SEQ ID NO: 1 or 3, of ananti-sense sequence of SEQ ID NO: 1 or 3, or of a naturally occurringallelic variant or mutant of SEQ ID NO:1 or 3. In one embodiment, anucleic acid molecule of the present invention comprises a nucleotidesequence which is greater than 50-100, 100-500, 500-1000, 1000-1500,1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500,4500-5000, 5000-5500, 5500-6000, 6000-6500, 6500-6700, or morenucleotides in length and hybridizes under stringent hybridizationconditions to a nucleic acid molecule of SEQ ID NO: 1 or 3.

Probes based on the MTP-1 nucleotide sequences can be used to detecttranscripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissue which misexpress an MTP-1 protein, such as by measuring a levelof an MTP-1-encoding nucleic acid in a sample of cells from a subjecte.g., detecting MTP-1 mRNA levels or determining whether a genomic MTP-1gene has been mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion of anMTP-1 protein” can be prepared by isolating a portion of the nucleotidesequence of SEQ ID NO: 1 or 3, which encodes a polypeptide having anMTP-1 biological activity (the biological activities of the MTP-1proteins are described herein), expressing the encoded portion of theMTP-1 protein (e.g., by recombinant expression in vitro) and assessingthe activity of the encoded portion of the MTP-1 protein.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO: 1 or 3, due todegeneracy of the genetic code and thus encode the same MTP-1 proteinsas those encoded by the nucleotide sequence shown in SEQ ID NO: 1 or 3.In another embodiment, an isolated nucleic acid molecule of theinvention has a nucleotide sequence encoding a protein having an aminoacid sequence shown in SEQ ID NO:2.

In addition to the MTP-1 nucleotide sequences shown in SEQ ID NO: 1 or3, it will be appreciated by those skilled in the art that DNA sequencepolymorphisms that lead to changes in the amino acid sequences of theMTP-1 proteins may exist within a population (e.g., the humanpopulation). Such genetic polymorphism in the MTP-1 genes may existamong individuals within a population due to natural allelic variation.As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules which include an open reading frame encoding an MTP-1protein, preferably a mammalian MTP-1 protein, and can further includenon-coding regulatory sequences, and introns.

Allelic variants of human MTP-1 include both functional andnon-functional MTP-1 proteins. Functional allelic variants are naturallyoccurring amino acid sequence variants of the human MTP-1 protein thatmaintain the ability to transport an MTP-1 substrate and/or modulatecellular homeostasis. Functional allelic variants will typically containonly conservative substitution of one or more amino acids of SEQ IDNO:2, or substitution, deletion or insertion of non-critical residues innon-critical regions of the protein.

Non-functional allelic variants are naturally occurring amino acidsequence variants of the human MTP-1 protein that do not have theability to bind or transport an MTP-1 substrate and/or carry out any ofthe MTP-1 activities described herein. Non-functional allelic variantswill typically contain a non-conservative substitution, a deletion, orinsertion or premature truncation of the amino acid sequence of SEQ IDNO:2, or a substitution, insertion or deletion in critical residues orcritical regions of the protein.

The present invention further provides non-human orthologues of thehuman MTP-1 protein. Orthologues of the human MTP-1 protein are proteinsthat are isolated from non-human organisms and possess the same MTP-1substrate binding and/or modulation of membrane excitability activitiesof the human MTP-1 protein. Orthologues of the human MTP-1 protein canreadily be identified as comprising an amino acid sequence that issubstantially identical to SEQ ID NO:2.

Moreover, nucleic acid molecules encoding other MTP-1 family membersand, thus, which have a nucleotide sequence which differs from the MTP-1sequences of SEQ ID NO: 1 or 3, are intended to be within the scope ofthe invention. For example, another MTP-1 cDNA can be identified basedon the nucleotide sequence of human MTP-1. Moreover, nucleic acidmolecules encoding MTP-1 proteins from different species, and which,thus, have a nucleotide sequence which differs from the MTP-1 sequencesof SEQ ID NO:1 or 3, are intended to be within the scope of theinvention. For example, a mouse MTP-1 cDNA can be identified based onthe nucleotide sequence of a human MTP-1.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the MTP-1 cDNAs of the invention can be isolated based ontheir homology to the MTP-1 nucleic acids disclosed herein using thecDNAs disclosed herein, or a portion thereof, as a hybridization probeaccording to standard hybridization techniques under stringenthybridization conditions. Nucleic acid molecules corresponding tonatural allelic variants and homologues of the MTP-1 cDNAs of theinvention can further be isolated by mapping to the same chromosome orlocus as the MTP-1 gene.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 15, 20, 25, 30 or more nucleotides in lengthand hybridizes under stringent conditions to the nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1 or 3. In otherembodiment, the nucleic acid is at least 50-100, 100-500, 500-1000,1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000,4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000-6500, 6500-6700, ormore nucleotides in length.

In one embodiment, an isolated nucleic acid molecule of the inventioncomprises the nucleotide sequence shown in SEQ ID NO:4 or 6. This cDNAmay comprise sequences encoding the human OAT4 protein (e.g., the“coding region”, from nucleotides 372-2021), as well as 5′ untranslatedsequence (nucleotides 1-371) and 3′ untranslated sequences (nucleotides2022-2206) of SEQ ID NO:4. Alternatively, the nucleic acid molecule cancomprise only the coding region of SEQ ID NO:4 (e.g., nucleotides372-2021, corresponding to SEQ ID NO:6). Accordingly, in anotherembodiment, an isolated nucleic acid molecule of the invention comprisesSEQ ID NO:6 and nucleotides 1-371 of SEQ ID NO:4. In yet anotherembodiment, the isolated nucleic acid molecule comprises SEQ ID NO:6 andnucleotides 2022-2206 of SEQ ID NO:4. In yet another embodiment, thenucleic acid molecule consists of the nucleotide sequence set forth asSEQ ID NO:4 or SEQ ID NO:6. In still another embodiment, the nucleicacid molecule can comprise the coding region of SEQ ID NO:4 (e.g.,nucleotides 372-2021, corresponding to SEQ ID NO:6), as well as a stopcodon (e.g., nucleotides 2022-2024 of SEQ ID NO:4). In anotherembodiment, the nucleic acid molecule comprises nucleotides 1-25 of SEQID NO:4 or nucleotides 2186-2206 of SEQ ID NO:4.

In another embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:7 or 9.This cDNA may comprise sequences encoding the human OAT4 protein (e.g.,the “coding region”, from nucleotides 104-2275), as well as 5′untranslated sequence (nucleotides 1-103) and 3′ untranslated sequences(nucleotides 2276-2634) of SEQ ID NO:7. Alternatively, the nucleic acidmolecule can comprise only the coding region of SEQ ID NO:7 (e.g.,nucleotides 104-2275, corresponding to SEQ ID NO:9). Accordingly, inanother embodiment, an isolated nucleic acid molecule of the inventioncomprises SEQ ID NO:9 and nucleotides 1-103 of SEQ ID NO:7. In yetanother embodiment, the isolated nucleic acid molecule comprises SEQ IDNO:9 and nucleotides 2276-2634 of SEQ ID NO:7. In yet anotherembodiment, the nucleic acid molecule consists of the nucleotidesequence set forth as SEQ ID NO:7 or SEQ ID NO:9. In still anotherembodiment, the nucleic acid molecule can comprise the coding region ofSEQ ID NO:7 (e.g., nucleotides 104-2275, corresponding to SEQ ID NO:9),as well as a stop codon (e.g., nucleotides 2276-2278 of SEQ ID NO:7). Inanother embodiment, the nucleic acid molecule comprises nucleotides1-1305, nucleotides 1622-2634, nucleotides 104-1305, or nucleotides1622-2275 of SEQ ID NO:7.

In still another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO:4, 6, 7, or 9, or a portion ofany of these nucleotide sequences. A nucleic acid molecule which iscomplementary to the nucleotide sequence shown in SEQ ID NO:4, 6, 7, or9, is one which is sufficiently complementary to the nucleotide sequenceshown in SEQ ID NO:4, 6, 7, or 9, such that it can hybridize to thenucleotide sequence shown in SEQ ID NO:4, 6, 7, or 9, thereby forming astable duplex.

In still another embodiment, an isolated nucleic acid molecule of thepresent invention comprises a nucleotide sequence which is at leastabout 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%,90%, 91%, 92%, 93%, 94%,95%, 96%,97%, 98%, 99%, 99.1% 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, 99.9% or more identical to the nucleotide sequence shown in SEQID NO:4, 6, 7, or 9 (e.g., to the entire length of the nucleotidesequence), or a portion or complement of any of these nucleotidesequences. In one embodiment, a nucleic acid molecule of the presentinvention comprises a nucleotide sequence which is at least (or nogreater than) 50, 100, 150, 200, 250, 300, 317, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200,1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1769,1800, 1850, 1869, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300,2350, 2400, 2450, 2500, 2550, 2600 or more nucleotides in length andhybridizes under stringent hybridization conditions to a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:4, 6, 7, or 9.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO:4, 6, 7, or 9, forexample, a fragment which can be used as a probe or primer or a fragmentencoding a portion of an OAT protein, e.g., a biologically activeportion of an OAT protein. The nucleotide sequence determined from thecloning of the OAT gene allows for the generation of probes and primersdesigned for use in identifying and/or cloning other OAT family members,as well as OAT homologues from other species. The probe/primer (e.g.,oligonucleotide) typically comprises substantially purifiedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12 or 15, preferably about 20 or 25, more preferably about30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sensesequence of SEQ ID NO:4, 6, 7, or 9, of an anti-sense sequence of SEQ IDNO:4, 6, 7, or 9, or of a naturally occurring allelic variant or mutantof SEQ ID NO:4, 6, 7, or 9.

Exemplary probes or primers are at least (or no greater than) 12, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more nucleotides inlength and/or comprise consecutive nucleotides of an isolated nucleicacid molecule described herein. Also included within the scope of thepresent invention are probes or primers comprising contiguous orconsecutive nucleotides of an isolated nucleic acid molecule describedherein, but for the difference of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 baseswithin the probe or primer sequence. Probes based on the OAT nucleotidesequences can be used to detect (e.g., specifically detect) transcriptsor genomic sequences encoding the same or homologous proteins. Inpreferred embodiments, the probe further comprises a label groupattached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. In anotherembodiment a set of primers is provided, e.g., primers suitable for usein a PCR, which can be used to amplify a selected region of an OATsequence, e.g., a domain, region, site or other sequence describedherein. The primers should be at least 5, 10, or 50 base pairs in lengthand less than 100, or less than 200, base pairs in length. The primersshould be identical, or differ by no greater than 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 bases when compared to a sequence disclosed herein or to thesequence of a naturally occurring variant. Such probes can be used as apart of a diagnostic test kit for identifying cells or tissue whichmisexpress an OAT protein, such as by measuring a level of anOAT-encoding nucleic acid in a sample of cells from a subject, e.g.,detecting OAT mRNA levels or determining whether a genomic OAT gene hasbeen mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion of anOAT protein” can be prepared by isolating a portion of the nucleotidesequence of SEQ ID NO:4, 6, 7, or 9, which encodes a polypeptide havingan OAT biological activity (the biological activities of the OATproteins are described herein), expressing the encoded portion of theOAT protein (e.g., by recombinant expression in vitro) and assessing theactivity of the encoded portion of the OAT protein. In an exemplaryembodiment, the nucleic acid molecule is at least 50, 100, 150, 200,250, 300, 317, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450,1500, 1550, 1600, 1650, 1700, 1750, 1769, 1800, 1850, 1869, 1900, 1950,2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550,2600 or more nucleotides in length and encodes a protein having an OATactivity (as described herein).

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:4, 6, 7, or 9, due todegeneracy of the genetic code and thus encode the same OAT proteins asthose encoded by the nucleotide sequence shown in SEQ ID NO:4, 6, 7, or9. In another embodiment, an isolated nucleic acid molecule of theinvention has a nucleotide sequence encoding a protein having an aminoacid sequence which differs by at least 1, but no greater than 5, 10,20, 50 or 100 amino acid residues from the amino acid sequence shown inSEQ ID NO:5 or 8. In yet another embodiment, the nucleic acid moleculeencodes the amino acid sequence of human OAT4 or OAT5. If an alignmentis needed for this comparison, the sequences should be aligned formaximum homology.

Nucleic acid variants can be naturally occurring, such as allelicvariants (same locus), homologues (different locus), and orthologues(different organism) or can be non naturally occurring. Non-naturallyoccurring variants can be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions (as compared in the encoded product).

Allelic variants result, for example, from DNA sequence polymorphismswithin a population (e.g., the human population) that lead to changes inthe amino acid sequences of the OAT proteins. Such genetic polymorphismin the OAT genes may exist among individuals within a population due tonatural allelic variation. As used herein, the terms “gene” and“recombinant gene” refer to nucleic acid molecules which include an openreading frame encoding an OAT protein, preferably a mammalian OATprotein, and can further include non-coding regulatory sequences, andintrons.

Accordingly, in one embodiment, the invention features isolated nucleicacid molecules which encode a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:5 or 8,wherein the nucleic acid molecule hybridizes to a complement of anucleic acid molecule comprising SEQ ID NO:4, 6, 7, or 9, for example,under stringent hybridization conditions.

Allelic variants of human OAT include both functional and non-functionalOAT proteins. Functional allelic variants are naturally occurring aminoacid sequence variants of the OAT protein that maintain the ability tobind an OAT substrate or target molecule, transport an OAT substrateacross a membrane, protect cells and/or tissues from organic anions,modulate inter- or intra-cellular signaling, and/or modulate hormoneresponses. Functional allelic variants will typically contain onlyconservative substitution of one or more amino acids of SEQ ID NO:5 or8, or substitution, deletion or insertion of non-critical residues innon-critical regions of the protein.

Non-functional allelic variants are naturally occurring amino acidsequence variants of the OAT proteins that, for example, do not have theability to bind an OAT substrate or target molecule, transport an OATsubstrate, protect cells and/or tissues from organic anions, modulateinter- or intra-cellular signaling, and/or modulate hormone responses.Non-functional allelic variants will typically contain anon-conservative substitution, a deletion, or insertion, or prematuretruncation of the amino acid sequence of SEQ ID NO:5 or 8, or asubstitution, insertion, or deletion in critical residues or criticalregions of the protein.

The present invention further provides non-human orthologues (e.g.,non-human orthologues of the human OAT4 or OAT5 proteins). Orthologuesof the human OAT proteins are proteins that are isolated from non-humanorganisms and possess the same OAT substrate-transporting mechanisms,substrate or target molecule binding mechanisms, mechanisms ofprotecting cells and/or tissues from organic anions, and/or inter- orintra-cellular signaling or hormonal modulating mechanisms of the humanOAT proteins. Orthologues of the human OAT proteins can readily beidentified as comprising an amino acid sequence that is substantiallyhomologous to SEQ ID NO:5 or 8.

Moreover, nucleic acid molecules encoding other OAT family members and,thus, which have a nucleotide sequence which differs from the OATsequences of SEQ ID NO:4, 6, 7, or 9, are intended to be within thescope of the invention. For example, another OAT cDNA can be identifiedbased on the nucleotide sequence of human OAT4 or OAT5. Moreover,nucleic acid molecules encoding OAT proteins from different species, andwhich, thus, have a nucleotide sequence which differs from the OATsequences of SEQ ID NO:4, 6, 7, or 9, are intended to be within thescope of the invention. For example, a mouse or monkey OAT cDNA can beidentified based on the nucleotide sequence of human OAT, e.g., OAT4 orOAT5.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the OAT cDNAs of the invention can be isolated based ontheir homology to the OAT nucleic acids disclosed herein using the cDNAsdisclosed herein, or a portion thereof, as a hybridization probeaccording to standard hybridization techniques under stringenthybridization conditions. Nucleic acid molecules corresponding tonatural allelic variants and homologues of the OAT cDNAs of theinvention can further be isolated by mapping to the same chromosome orlocus as the OAT gene.

Orthologues, homologues and allelic variants can be identified usingmethods known in the art (e.g., by hybridization to an isolated nucleicacid molecule of the present invention, for example, under stringenthybridization conditions). In one embodiment, an isolated nucleic acidmolecule of the invention is at least 15, 20, 25, 30 or more nucleotidesin length and hybridizes under stringent conditions to the nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:4, 6, 7, or 9.In other embodiment, the nucleic acid is at least 50, 100, 150, 200,250, 300, 317, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450,1500, 1550, 1600, 1650, 1700, 1750, 1769, 1800, 1850, 1869, 1900, 1950,2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550,2600 or more nucleotides in length.

In one embodiment, an isolated nucleic acid molecule of the inventioncomprises the nucleotide sequence shown in SEQ ID NO: 12. The sequenceof SEQ ID NO: 12 corresponds to the human HST-1 cDNA. This cDNAcomprises sequences encoding the human HST-1 polypeptide (i.e., “thecoding region”, from nucleotides 13-1732) as well as 5′ untranslatedsequences (nucleotides 1-12) and 3′ untranslated sequences (nucleotides1733-1917). Alternatively, the nucleic acid molecule can comprise onlythe coding region of SEQ ID NO:12 (e.g., nucleotides 13-1732,corresponding to SEQ ID NO: 14). Accordingly, in another embodiment, theisolated nucleic acid molecule comprises SEQ ID NO: 14 and nucleotides1-12 and 1733-1917 of SEQ ID NO:12. In yet another embodiment, thenucleic acid molecule consists of the nucleotide sequence set forth asSEQ ID NO:12 or 14.

In still another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO:12 or 14, or a portion of any ofthese nucleotide sequences. A nucleic acid molecule which iscomplementary to the nucleotide sequence shown in SEQ ID NO:12 or 14, isone which is sufficiently complementary to the nucleotide sequence shownin SEQ ID NO:12 or 14, such that it can hybridize to the nucleotidesequence shown in SEQ ID NO:12 or 14, thereby forming a stable duplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the present invention comprises a nucleotide sequence which is atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to the nucleotidesequence shown in SEQ ID NO:12 or 14 (e.g., to the entire length of thenucleotide sequence), or a portion of any of these nucleotide sequences.In one embodiment, a nucleic acid molecule of the present inventioncomprises a nucleotide sequence which is at least (or no greater than)50, 57, 63, 72, 100, 124, 150, 172, 175, 200, 250, 268, 300, 305, 328,350, 400, 431, 450, 495, 500, 550, 600, 650, 700, 750, 800, 804, 850,900, 950, 1000, 1050, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550,1600, 1650, 1700, 1750, 1800, 1850, 1900 or more nucleotides in lengthand hybridizes under stringent hybridization conditions to a complementof a nucleic acid molecule of SEQ ID NO: 12 or 14.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO: 12 or 14, forexample, a fragment which can be used as a probe or primer or a fragmentencoding a portion of an HST-1 polypeptide, e.g., a biologically activeportion of an HST-1 polypeptide. The nucleotide sequence determined fromthe cloning of the HST-1 gene allows for the generation of probes andprimers designed for use in identifying and/or cloning other HST-1family members, as well as HST-1 homologues from other species. Theprobe/primer typically comprises substantially purified oligonucleotide.The probe/primer (e.g., oligonucleotide) typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12 or 15, preferably about 20 or 25, more preferably about30, 35, 40, 45, 50, 55, 60, 65, 75, 80, 85, 90, 95, or 100 or moreconsecutive nucleotides of a sense sequence of SEQ ID NO: 12 or 14, ofan anti-sense sequence of SEQ ID NO: 12 or 14, or of a naturallyoccurring allelic variant or mutant of SEQ ID NO:12 or 14.

Exemplary probes or primers are at least 12, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75 or more nucleotides in length and/or compriseconsecutive nucleotides of an isolated nucleic acid molecule describedherein. Probes based on the HST-1 nucleotide sequences can be used todetect (e.g., specifically detect) transcripts or genomic sequencesencoding the same or homologous polypeptides. In preferred embodiments,the probe further comprises a label group attached thereto, e.g., thelabel group can be a radioisotope, a fluorescent compound, an enzyme, oran enzyme co-factor. In another embodiment a set of primers is provided,e.g., primers suitable for use in a PCR, which can be used to amplify aselected region of an HST-1 sequence, e.g., a domain, region, site orother sequence described herein. The primers should be at least 5, 10,20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides in length. Suchprobes can be used as a part of a diagnostic test kit for identifyingcells or tissue which misexpress an HST-1 polypeptide, such as bymeasuring a level of an HST-1-encoding nucleic acid in a sample of cellsfrom a subject e.g., detecting HST-1 mRNA levels or determining whethera genomic HST-1 gene has been mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion of anHST-1 polypeptide” can be prepared by isolating a portion of thenucleotide sequence of SEQ ID NO:12 or 14, which encodes a polypeptidehaving an HST-1 biological activity (the biological activities of theHST-1 polypeptides are described herein), expressing the encoded portionof the HST-1 polypeptide (e.g., by recombinant expression in vitro) andassessing the activity of the encoded portion of the HST-1 polypeptide.In an exemplary embodiment, the nucleic acid molecule is at least 50,57, 63, 72, 100, 124, 150, 172, 175, 200, 250, 268, 300, 305, 328, 350,400, 431, 450, 495, 500, 550, 600, 650, 700, 750, 800, 804, 850, 900,950, 1000, 1050, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600,1650, 1700, 1750, 1800, 1850, 1900 or more nucleotides in length andencodes a polypeptide having an HST-1 activity (as described herein).

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:12 or 14. Suchdifferences can be due to due to degeneracy of the genetic code, thusresulting in a nucleic acid which encodes the same HST-1 polypeptides asthose encoded by the nucleotide sequence shown in SEQ ID NO:12 or 14. Inanother embodiment, an isolated nucleic acid molecule of the inventionhas a nucleotide sequence encoding a polypeptide having an amino acidsequence which differs by at least 1, but no greater than 5, 10, 20, 50,100, 150, 155, 200, 250, 300, 350, 350, 400, 450, or 500 amino acidresidues from the amino acid sequence shown in SEQ ID NO: 13. In yetanother embodiment, the nucleic acid molecule encodes the amino acidsequence of human HST-1. If an alignment is needed for this comparison,the sequences should be aligned for maximum homology.

Nucleic acid variants can be naturally occurring, such as allelicvariants (same locus), homologues (different locus), and orthologues(different organism) or can be non naturally occurring. Non-naturallyoccurring variants can be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions (as compared in the encoded product).

Allelic variants result, for example, from DNA sequence polymorphismswithin a population (e.g., the human population) that lead to changes inthe amino acid sequences of the HST-1 polypeptides. Such geneticpolymorphism in the HST-1 genes may exist among individuals within apopulation due to natural allelic variation. As used herein, the terms“gene” and “recombinant gene” refer to nucleic acid molecules whichinclude an open reading frame encoding an HST-1 polypeptide, preferablya mammalian HST-1 polypeptide, and can further include non-codingregulatory sequences, and introns.

Accordingly, in one embodiment, the invention features isolated nucleicacid molecules which encode a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO: 13, whereinthe nucleic acid molecule hybridizes to a complement of a nucleic acidmolecule comprising SEQ ID NO:12 or 14, for example, under stringenthybridization conditions.

Allelic variants of human HST-1 include both functional andnon-functional HST-1 polypeptides. Functional allelic variants arenaturally occurring amino acid sequence variants of the human HST-1polypeptide that have an HST-1 activity, e.g., maintain the ability tobind an HST-1 ligand or substrate and/or modulate sugar transport, orsugar homeostasis. Functional allelic variants will typically containonly conservative substitution of one or more amino acids of SEQ ID NO:13, or substitution, deletion or insertion of non-critical residues innon-critical regions of the polypeptide.

Non-functional allelic variants are naturally occurring amino acidsequence variants of the human HST-1 polypeptide that do not have anHST-1 activity, e.g., they do not have the ability to transport sugarsinto and out of cells or to modulate sugar homeostasis. Non-functionalallelic variants will typically contain a non-conservative substitution,a deletion, or insertion or premature truncation of the amino acidsequence of SEQ ID NO: 13, or a substitution, insertion or deletion incritical residues or critical regions.

The present invention further provides non-human orthologues of thehuman HST-1 polypeptide. Orthologues of human HST-1 polypeptides arepolypeptides that are isolated from non-human organisms and possess thesame HST-1 activity, e.g., ligand binding and/or modulation of sugartransport mechanisms, as the human HST-1 polypeptide. Orthologues of thehuman HST-1 polypeptide can readily be identified as comprising an aminoacid sequence that is substantially identical to SEQ ID NO:13.

Moreover, nucleic acid molecules encoding other HST-1 family membersand, thus, which have a nucleotide sequence which differs from the HST-1sequences of SEQ ID NO:12 or 14, are intended to be within the scope ofthe invention. For example, another HST-1 cDNA can be identified basedon the nucleotide sequence of human HST-1. Moreover, nucleic acidmolecules encoding HST-1 polypeptides from different species, and which,thus, have a nucleotide sequence which differs from the HST-1 sequencesof SEQ ID NO:12 or 14, are intended to be within the scope of theinvention. For example, a mouse HST-1 cDNA can be identified based onthe nucleotide sequence of a human HST-1.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the HST-1 cDNAs of the invention can be isolated based ontheir homology to the HST-1 nucleic acids disclosed herein using thecDNAs disclosed herein, or a portion thereof, as a hybridization probeaccording to standard hybridization techniques under stringenthybridization conditions. Nucleic acid molecules corresponding tonatural allelic variants and homologues of the HST-1 cDNAs of theinvention can further be isolated by mapping to the same chromosome orlocus as the HST-1 gene.

Orthologues, homologues and allelic variants can be identified usingmethods known in the art (e.g., by hybridization to an isolated nucleicacid molecule of the present invention, for example, under stringenthybridization conditions). In one embodiment, an isolated nucleic acidmolecule of the invention is at least 15, 20, 25, 30 or more nucleotidesin length and hybridizes under stringent conditions to the nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO: 12 or 14. Inother embodiment, the nucleic acid is at least 50, 57, 63, 72, 100, 124,150, 172, 175, 200, 250, 268, 300, 305, 328, 350, 400, 431, 450, 495,500, 550, 600, 650, 700, 750, 800, 804, 850, 900, 950, 1000, 1050, 1200,1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800,1850, 1900 or more nucleotides in length.

In one embodiment, an isolated nucleic acid molecule of the inventioncomprises the nucleotide sequence shown in SEQ ID NO:15. The sequence ofSEQ ID NO:15 corresponds to the human TP-2 cDNA. This cDNA comprisessequences encoding the human TP-2 polypeptide (i.e., “the codingregion”, from nucleotides 67-1491) as well as 5′ untranslated sequences(nucleotides 1-66) and 3′ untranslated sequences (nucleotides1492-1963). Alternatively, the nucleic acid molecule can comprise onlythe coding region of SEQ ID NO:15 (e.g., nucleotides 67-1491,corresponding to SEQ ID NO:17). Accordingly, in another embodiment, theisolated nucleic acid molecule comprises SEQ ID NO: 17 and nucleotides1-66 and 1492-1963 of SEQ ID NO: 15. In yet another embodiment, thenucleic acid molecule consists of the nucleotide sequence set forth asSEQ ID NO: 15 or 17.

In still another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO: 15 or 17, or a portion of any ofthese nucleotide sequences. A nucleic acid molecule which iscomplementary to the nucleotide sequence shown in SEQ ID NO:15 or 17, isone which is sufficiently complementary to the nucleotide sequence shownin SEQ ID NO:15 or 17, such that it can hybridize to the nucleotidesequence shown in SEQ ID NO:15 or 17, thereby forming a stable duplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the present invention comprises a nucleotide sequence which is atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or more identical to the nucleotide sequence shown in SEQ IDNO:15 or 17 (e.g., to the entire length of the nucleotide sequence), ora portion of any of these nucleotide sequences. In one embodiment, anucleic acid molecule of the present invention comprises a nucleotidesequence which is at least (or no greater than) 50, 100, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 1950 or morenucleotides in length and hybridizes under stringent hybridizationconditions to a complement of a nucleic acid molecule of SEQ ID NO: 15or 17.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO: 15 or 17, forexample, a fragment which can be used as a probe or primer or a fragmentencoding a portion of a TP-2 polypeptide, e.g., a biologically activeportion of a TP-2 polypeptide. The nucleotide sequence determined fromthe cloning of the TP-2 gene allows for the generation of probes andprimers designed for use in identifying and/or cloning other TP-2 familymembers, as well as TP-2 homologues from other species. The probe/primertypically comprises substantially purified oligonucleotide. Theprobe/primer (e.g., oligonucleotide) typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12 or 15, preferably about 20 or 25, more preferably about30, 35, 40, 45, 50, 55, 60, 65, 75, 80, 85, 90, 95, or 100 or moreconsecutive nucleotides of a sense sequence of SEQ ID NO: 15 or 17, ofan anti-sense sequence of SEQ ID NO: 15 or 17, or of a naturallyoccurring allelic variant or mutant of SEQ ID NO: 15 or 17.

Exemplary probes or primers are at least 12, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75 or more nucleotides in length and/or compriseconsecutive nucleotides of an isolated nucleic acid molecule describedherein. Probes based on the TP-2 nucleotide sequences can be used todetect (e.g., specifically detect) transcripts or genomic sequencesencoding the same or homologous polypeptides. In preferred embodiments,the probe further comprises a label group attached thereto, e.g., thelabel group can be a radioisotope, a fluorescent compound, an enzyme, oran enzyme co-factor. In another embodiment a set of primers is provided,e.g., primers suitable for use in a PCR, which can be used to amplify aselected region of a TP-2 sequence, e.g., a domain, region, site orother sequence described herein. The primers should be at least 5, 10,20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides in length. Suchprobes can be used as a part of a diagnostic test kit for identifyingcells or tissue which misexpress a TP-2 polypeptide, such as bymeasuring a level of a TP-2-encoding nucleic acid in a sample of cellsfrom a subject e.g., detecting TP-2 mRNA levels or determining whether agenomic TP-2 gene has been mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion of aTP-2 polypeptide” can be prepared by isolating a portion of thenucleotide sequence of SEQ ID NO:15 or 17, which encodes a polypeptidehaving a TP-2 biological activity (the biological activities of the TP-2polypeptides are described herein), expressing the encoded portion ofthe TP-2 polypeptide (e.g., by recombinant expression in vitro) andassessing the activity of the encoded portion of the TP-2 polypeptide.In an exemplary embodiment, the nucleic acid molecule is at least 50,100, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800,1900, 1950 or more nucleotides in length and encodes a polypeptidehaving a TP-2 activity (as described herein).

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:15 or 17. Suchdifferences can be due to due to degeneracy of the genetic code, thusresulting in a nucleic acid which encodes the same TP-2 polypeptides asthose encoded by the nucleotide sequence shown in SEQ ID NO:15 or 17. Inanother embodiment, an isolated nucleic acid molecule of the inventionhas a nucleotide sequence encoding a polypeptide having an amino acidsequence which differs by at least 1, but no greater than 5, 10, 20, 50or 100 amino acid residues from the amino acid sequence shown in SEQ IDNO: 16. In yet another embodiment, the nucleic acid molecule encodes theamino acid sequence of human TP-2. If an alignment is needed for thiscomparison, the sequences should be aligned for maximum homology.

Nucleic acid variants can be naturally occurring, such as allelicvariants (same locus), homologues (different locus), and orthologues(different organism) or can be non naturally occurring. Non-naturallyoccurring variants can be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions (as compared in the encoded product).

Allelic variants result, for example, from DNA sequence polymorphismswithin a population (e.g., the human population) that lead to changes inthe amino acid sequences of the TP-2 polypeptides. Such geneticpolymorphism in the TP-2 genes may exist among individuals within apopulation due to natural allelic variation. As used herein, the terms“gene” and “recombinant gene” refer to nucleic acid molecules whichinclude an open reading frame encoding a TP-2 polypeptide, preferably amammalian TP-2 polypeptide, and can further include non-codingregulatory sequences, and introns.

Accordingly, in one embodiment, the invention features isolated nucleicacid molecules which encode a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:16, whereinthe nucleic acid molecule hybridizes to a complement of a nucleic acidmolecule comprising SEQ ID NO:15 or 17, for example, under stringenthybridization conditions.

Allelic variants of human TP-2 include both functional andnon-functional TP-2 polypeptides. Functional allelic variants arenaturally occurring amino acid sequence variants of the human TP-2polypeptide that have a TP-2 activity, e.g., maintain the ability tobind a TP-2 ligand or substrate and/or modulate the import and export ofmolecules from cells or across membranes, e.g., monosaccharides.Functional allelic variants will typically contain only conservativesubstitution of one or more amino acids of SEQ ID NO: 16, orsubstitution, deletion or insertion of non-critical residues innon-critical regions of the polypeptide.

Non-functional allelic variants are naturally occurring amino acidsequence variants of the human TP-2 polypeptide that do not have a TP-2activity, e.g., they do not have the ability to transport molecules intoand out of cells or across membranes. Non-functional allelic variantswill typically contain a non-conservative substitution, a deletion, orinsertion or premature truncation of the amino acid sequence of SEQ IDNO: 16, or a substitution, insertion or deletion in critical residues orcritical regions.

The present invention further provides non-human orthologues of thehuman TP-2 polypeptide. Orthologues of human TP-2 polypeptides arepolypeptides that are isolated from non-human organisms and possess thesame TP-2 activity, e.g., ligand binding and/or modulation of import andexport of molecules from cells or across membranes, e.g.,monosaccharides, as the human TP-2 polypeptide. Orthologues of the humanTP-2 polypeptide can readily be identified as comprising an amino acidsequence that is substantially identical to SEQ ID NO:16.

Moreover, nucleic acid molecules encoding other TP-2 family members and,thus, which have a nucleotide sequence which differs from the TP-2sequences of SEQ ID NO:15 or 17, are intended to be within the scope ofthe invention. For example, another TP-2 cDNA can be identified based onthe nucleotide sequence of human TP-2. Moreover, nucleic acid moleculesencoding TP-2 polypeptides from different species, and which, thus, havea nucleotide sequence which differs from the TP-2 sequences of SEQ IDNO: 15 or 17, are intended to be within the scope of the invention. Forexample, a mouse TP-2 cDNA can be identified based on the nucleotidesequence of a human TP-2.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the TP-2 cDNAs of the invention can be isolated based ontheir homology to the TP-2 nucleic acids disclosed herein using thecDNAs disclosed herein, or a portion thereof, as a hybridization probeaccording to standard hybridization techniques under stringenthybridization conditions. Nucleic acid molecules corresponding tonatural allelic variants and homologues of the TP-2 cDNAs of theinvention can further be isolated by mapping to the same chromosome orlocus as the TP-2 gene.

Orthologues, homologues and allelic variants can be identified usingmethods known in the art (e.g., by hybridization to an isolated nucleicacid molecule of the present invention, for example, under stringenthybridization conditions). In one embodiment, an isolated nucleic acidmolecule of the invention is at least 15, 20, 25, 30 or more nucleotidesin length and hybridizes under stringent conditions to the nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO: 15 or 17. Inother embodiment, the nucleic acid is at least 50, 100, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 1950 or morenucleotides in length.

In one embodiment, an isolated nucleic acid molecule of the inventioncomprises the nucleotide sequence shown in SEQ ID NO:19 or 21. This cDNAmay comprise sequences encoding the human PLTR-1 protein (e.g., the“coding region”, from nucleotides 171-3740), as well as 5′ untranslatedsequence (nucleotides 1-170) and 3′ untranslated sequences (nucleotides3741-4693) of SEQ ID NO:19. Alternatively, the nucleic acid molecule cancomprise only the coding region of SEQ ID NO:19 (e.g., nucleotides171-3740, corresponding to SEQ ID NO:21). Accordingly, in anotherembodiment, an isolated nucleic acid molecule of the invention comprisesSEQ ID NO:21 and nucleotides 1-170 of SEQ ID NO:19. In yet anotherembodiment, the isolated nucleic acid molecule comprises SEQ ID NO:21and nucleotides 3741-4693 of SEQ ID NO: 19. In yet another embodiment,the nucleic acid molecule consists of the nucleotide sequence set forthas SEQ ID NO:19 or 21. In another embodiment, the nucleic acid moleculecan comprise the coding region of SEQ ID NO:19 (e.g., nucleotides171-3740, corresponding to SEQ ID NO:21), as well as a stop codon (e.g.,nucleotides 3741-3743 of SEQ ID NO: 19). In other embodiments, thenucleic acid molecule can comprise nucleotides 1-743 of SEQ ID NO: 19.

In still another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO: 19 or 21, or a portion of any ofthese nucleotide sequences. A nucleic acid molecule which iscomplementary to the nucleotide sequence shown in SEQ ID NO: 19 or 21,is one which is sufficiently complementary to the nucleotide sequenceshown in SEQ ID NO:19 or 21, such that it can hybridize to thenucleotide sequence shown in SEQ ID NO:19 or 21, thereby forming astable duplex.

In still another embodiment, an isolated nucleic acid molecule of thepresent invention comprises a nucleotide sequence which is at leastabout 75%, 79%, 80%, 81%, 85%, 90%, 91%,92%,93%, 94%, 95%, 96%, 97%,98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or more identical to the nucleotide sequence shown in SEQ ID NO: 19 or21 (e.g., to the entire length of the nucleotide sequence), or a portionor complement of any of these nucleotide sequences. In one embodiment, anucleic acid molecule of the present invention comprises a nucleotidesequence which is at least (or no greater than) 50, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 676, 677, 689, 690, 691, 692,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300,1350, 1400, 1450, 1500, 1550, 1562, 1600, 1610, 1660, 1700, 1750, 1800,1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2373,2374, 2375, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850,2900, 2950, 3000, 3050, 3063, 3064, 3100, 3150, 3200, 3250, 3300, 3350,3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3753, 3754, 3800, 3850,3900, 3950, 4000, 4050, 4100, 4150, 4200, 4250, 4300, 4350, 4400, 4450,4500, 4550, 4600, 4650 or more nucleotides in length and hybridizesunder stringent hybridization conditions to a complement of a nucleicacid molecule of SEQ ID NO:19 or 21.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO: 19 or 21, forexample, a fragment which can be used as a probe or primer or a fragmentencoding a portion of a PLTR-1 protein, e.g., a biologically activeportion of a PLTR-1 protein. The nucleotide sequence determined from thecloning of the PLTR-1 gene allows for the generation of probes andprimers designed for use in identifying and/or cloning other PLTR-1family members, as well as PLTR-1 homologues from other species. Theprobe/primer (e.g., oligonucleotide) typically comprises substantiallypurified oligonucleotide. The oligonucleotide typically comprises aregion of nucleotide sequence that hybridizes under stringent conditionsto at least about 12 or 15, preferably about 20 or 25, more preferablyabout 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of asense sequence of SEQ ID NO: 19 or 21, of an anti-sense sequence of SEQID NO:19 or 21, or of a naturally occurring allelic variant or mutant ofSEQ ID NO:19 or 21.

Exemplary probes or primers are at least (or no greater than) 12 or 15,20 or 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more nucleotides inlength and/or comprise consecutive nucleotides of an isolated nucleicacid molecule described herein. Also included within the scope of thepresent invention are probes or primers comprising contiguous orconsecutive nucleotides of an isolated nucleic acid molecule describedherein, but for the difference of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 baseswithin the probe or primer sequence. Probes based on the PLTR-1nucleotide sequences can be used to detect (e.g., specifically detect)transcripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. In anotherembodiment a set of primers is provided, e.g., primers suitable for usein a PCR, which can be used to amplify a selected region of a PLTR-1sequence, e.g., a domain, region, site or other sequence describedherein. The primers should be at least 5, 10, or 50 base pairs in lengthand less than 100, or less than 200, base pairs in length. The primersshould be identical, or differ by no greater than 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 bases when compared to a sequence disclosed herein or to thesequence of a naturally occurring variant. Such probes can be used as apart of a diagnostic test kit for identifying cells or tissue whichmisexpress a PLTR-1 protein, such as by measuring a level of aPLTR-1-encoding nucleic acid in a sample of cells from a subject, e.g.,detecting PLTR-1 mRNA levels or determining whether a genomic PLTR-1gene has been mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion of aPLTR-1 protein” can be prepared by isolating a portion of the nucleotidesequence of SEQ ID NO:19 or 21, which encodes a polypeptide having aPLTR-1 biological activity (the biological activities of the PLTR-1proteins are described herein), expressing the encoded portion of thePLTR-1 protein (e.g., by recombinant expression in vitro) and assessingthe activity of the encoded portion of the PLTR-1 protein. In anexemplary embodiment, the nucleic acid molecule is at least 50, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 676, 677, 689,690, 691, 692, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1562, 1600, 1610, 1660,1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250,2300, 2350, 2373, 2374, 2375, 2400, 2450, 2500, 2550, 2600, 2650, 2700,2750, 2800, 2850, 2900, 2950, 3000, 3050, 3063, 3064, 3100, 3150, 3200,3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3753,3754, 3800, 3850, 3900, 3950, 4000, 4050, 4100, 4150, 4200, 4250, 4300,4350, 4400, 4450, 4500, 4550, 4600, 4650 or more nucleotides in lengthand encodes a protein having a PLTR-1 activity (as described herein).

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO: 19 or 21, due todegeneracy of the genetic code and thus encode the same PLTR-1 proteinsas those encoded by the nucleotide sequence shown in SEQ ID NO: 19 or21. In another embodiment, an isolated nucleic acid molecule of theinvention has a nucleotide sequence encoding a protein having an aminoacid sequence which differs by at least 1, but no greater than 5, 10,20, 50 or 100 amino acid residues from the amino acid sequence shown inSEQ ID NO:20. In yet another embodiment, the nucleic acid moleculeencodes the amino acid sequence of human PLTR-1. If an alignment isneeded for this comparison, the sequences should be aligned for maximumhomology.

Nucleic acid variants can be naturally occurring, such as allelicvariants (same locus), homologues (different locus), and orthologues(different organism) or can be non naturally occurring. Non-naturallyoccurring variants can be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions (as compared in the encoded product).

Allelic variants result, for example, from DNA sequence polymorphismswithin a population (e.g., the human population) that lead to changes inthe amino acid sequences of the PLTR-1 proteins. Such geneticpolymorphism in the PLTR-1 genes may exist among individuals within apopulation due to natural allelic variation. As used herein, the terms“gene” and “recombinant gene” refer to nucleic acid molecules whichinclude an open reading frame encoding a PLTR-1 protein, preferably amammalian PLTR-1 protein, and can further include non-coding regulatorysequences, and introns.

Accordingly, in one embodiment, the invention features isolated nucleicacid molecules which encode a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:20, whereinthe nucleic acid molecule hybridizes to a complement of a nucleic acidmolecule comprising SEQ ID NO: 19 or 21, for example, under stringenthybridization conditions.

Allelic variants of PLTR-1, e.g., human PLTR-1, include both functionaland non-functional PLTR-1 proteins. Functional allelic variants arenaturally occurring amino acid sequence variants of the PLTR-1 proteinthat maintain the ability to, e.g., bind or interact with a PLTR-1substrate or target molecule, transport a PLTR-1 substrate or targetmolecule (e.g., a phospholipid) across a cellular membrane, hydrolyzeATP, be phosphorylated or dephosphorylated, adopt an E1 conformation oran E2 conformation, and/or modulate cellular signaling, growth,proliferation, differentiation, absorption, or secretion. Functionalallelic variants will typically contain only conservative substitutionof one or more amino acids of SEQ ID NO:20, or substitution, deletion orinsertion of non-critical residues in non-critical regions of theprotein.

Non-functional allelic variants are naturally occurring amino acidsequence variants of the PLTR-1 protein, e.g., human PLTR-1, that do nothave the ability to, e.g., bind or interact with a PLTR-1 substrate ortarget molecule, transport a PLTR-1 substrate or target molecule (e.g.,a phospholipid) across a cellular membrane, hydrolyze ATP, bephosphorylated or dephosphorylated, adopt an E1 conformation or an E2conformation, and/or modulate cellular signaling, growth, proliferation,differentiation, absorption, or secretion. Non-functional allelicvariants will typically contain a non-conservative substitution, adeletion, or insertion, or premature truncation of the amino acidsequence of SEQ ID NO:20, or a substitution, insertion, or deletion incritical residues or critical regions of the protein.

The present invention further provides non-human orthologues (e.g.,non-human orthologues of the human PLTR-1 protein). Orthologues of thehuman PLTR-1 protein are proteins that are isolated from non-humanorganisms and possess the same PLTR-1 substrate or target moleculebinding mechanisms, phospholipid transporting activity, ATPase activity,and/or modulation of cellular signaling mechanisms of the human PLTR-1proteins. Orthologues of the human PLTR-1 protein can readily beidentified as comprising an amino acid sequence that is substantiallyhomologous to SEQ ID NO:20.

Moreover, nucleic acid molecules encoding other PLTR-1 family membersand, thus, which have a nucleotide sequence which differs from thePLTR-1 sequences of SEQ ID NO: 19 or 21, are intended to be within thescope of the invention. For example, another PLTR-1 cDNA can beidentified based on the nucleotide sequence of human PLTR-1. Moreover,nucleic acid molecules encoding PLTR-1 proteins from different species,and which, thus, have a nucleotide sequence which differs from thePLTR-1 sequences of SEQ ID NO: 19 or 21, are intended to be within thescope of the invention. For example, a mouse or monkey PLTR-1 cDNA canbe identified based on the nucleotide sequence of a human PLTR-1.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the PLTR-1 cDNAs of the invention can be isolated based ontheir homology to the PLTR-1 nucleic acids disclosed herein using thecDNAs disclosed herein, or a portion thereof, as a hybridization probeaccording to standard hybridization techniques under stringenthybridization conditions. Nucleic acid molecules corresponding tonatural allelic variants and homologues of the PLTR-1 cDNAs of theinvention can further be isolated by mapping to the same chromosome orlocus as the PLTR-1 gene.

Orthologues, homologues and allelic variants can be identified usingmethods known in the art (e.g., by hybridization to an isolated nucleicacid molecule of the present invention, for example, under stringenthybridization conditions). In one embodiment, an isolated nucleic acidmolecule of the invention is at least 15, 20, 25, 30 or more nucleotidesin length and hybridizes under stringent conditions to the nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO: 19 or 21. Inother embodiment, the nucleic acid is at least 50, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 676, 677, 689, 690, 691, 692,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300,1350, 1400, 1450, 1500, 1550, 1562, 1600, 1610, 1660, 1700, 1750, 1800,1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2373,2374, 2375, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850,2900, 2950, 3000, 3050, 3063, 3064, 3100, 3150, 3200, 3250, 3300, 3350,3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3753, 3754, 3800, 3850,3900, 3950, 4000, 4050, 4100, 4150, 4200, 4250, 4300, 4350, 4400, 4450,4500, 4550, 4600, 4650 or more nucleotides in length.

In one embodiment, an isolated nucleic acid molecule of the inventioncomprises the nucleotide sequence shown in SEQ ID NO:27. The sequence ofSEQ ID NO:27 corresponds to the human TFM-2 cDNA. This cDNA comprisessequences encoding the human TFM-2 polypeptide (i.e., “the codingregion”, from nucleotides 615-1794) as well as 5′ untranslated sequences(nucleotides 1-614) and 3′ untranslated sequences (nucleotides1795-3524). Alternatively, the nucleic acid molecule can comprise onlythe coding region of SEQ ID NO:27 (e.g., nucleotides 615-1794,corresponding to SEQ ID NO:29). Accordingly, in another embodiment, theisolated nucleic acid molecule comprises SEQ ID NO:29 and nucleotides1-614 and 1795-3524 of SEQ ID NO:27. In yet another embodiment, thenucleic acid molecule consists of the nucleotide sequence set forth asSEQ ID NO:27 or SEQ ID NO:29.

In another embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:30. Thesequence of SEQ ID NO:30 corresponds to the human TFM-3 cDNA. This cDNAcomprises sequences encoding the human TFM-3 polypeptide (i.e., “thecoding region”, from nucleotides 384-1602) as well as 5′ untranslatedsequences (nucleotides 1-383) and 3′ untranslated sequences (nucleotides1603-1855). Alternatively, the nucleic acid molecule can comprise onlythe coding region of SEQ ID NO:30 (e.g., nucleotides 384-1602,corresponding to SEQ ID NO:32). Accordingly, in another embodiment, theisolated nucleic acid molecule comprises SEQ ID NO:32 and nucleotides1-383 and 1603-1855 of SEQ ID NO:30. In yet another embodiment, thenucleic acid molecule consists of the nucleotide sequence set forth asSEQ ID NO:30 or SEQ ID NO:32.

In still another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO:27, 29, 30, or 32, or a portionof any of these nucleotide sequences. A nucleic acid molecule which iscomplementary to the nucleotide sequence shown in SEQ ID NO:27, 29, 30,or 32, is one which is sufficiently complementary to the nucleotidesequence shown in SEQ ID NO:27, 29, 30, or 32, such that it canhybridize to the nucleotide sequence shown in SEQ ID NO:27, 29, 30, or32, thereby forming a stable duplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the present invention comprises a nucleotide sequence which is atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or more identical to the nucleotide sequence shown in SEQ IDNO:27, 29, 30, or 32 (e.g., to the entire length of the nucleotidesequence), or a portion of any of these nucleotide sequences. In oneembodiment, a nucleic acid molecule of the present invention comprises anucleotide sequence which is at least (or no greater than) 50-100,100-250, 250-500, 500-750, 750-1000, 1000-1250, 1250-1500, 1500-1750,1750-2000, 2000-2250, 2250-2500, 2500-2750, 2750-3000, 3000-3250,3250-3500 or more nucleotides in length and hybridizes under stringenthybridization conditions to a complement of a nucleic acid molecule ofSEQ ID NO:27 or 29. In another embodiment, a nucleic acid molecule ofthe present invention comprises a nucleotide sequence which is at least(or no greater than) 50-100, 100-250, 250-500, 500-750, 750-1000,1000-1250, 1250-1500, 1500-1750, 1750-1850 or more nucleotides in lengthand hybridizes under stringent hybridization conditions to a complementof a nucleic acid molecule of SEQ ID NO:30 or 32.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO:27, 29, 30, or 32, forexample, a fragment which can be used as a probe or primer or a fragmentencoding a portion of a TFM-2 and/or TFM-3 polypeptide, e.g., abiologically active portion of a TFM-2 and/or TFM-3 polypeptide. Thenucleotide sequence determined from the cloning of the TFM-2 and/orTFM-3 gene allows for the generation of probes and primers designed foruse in identifying and/or cloning other TFM-2 and/or TFM-3 familymembers, as well as TFM-2 and/or TFM-3 homologues from other species.The probe/primer typically comprises substantially purifiedoligonucleotide. The probe/primer (e.g., oligonucleotide) typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12 or 15, preferably about 20 or25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, 75, 80, 85,90, 95, or 100 or more consecutive nucleotides of a sense sequence ofSEQ ID NO:27, 29, 30, or 32, of an anti-sense sequence of SEQ ID NO:27,29, 30, or 32, or of a naturally occurring allelic variant or mutant ofSEQ ID NO:27, 29, 30, or 32.

Exemplary probes or primers are at least 12, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75 or more nucleotides in length and/or compriseconsecutive nucleotides of an isolated nucleic acid molecule describedherein. Probes based on the TFM-2 and/or TFM-3 nucleotide sequences canbe used to detect (e.g., specifically detect) transcripts or genomicsequences encoding the same or homologous polypeptides. In preferredembodiments, the probe further comprises a label group attached thereto,e.g., the label group can be a radioisotope, a fluorescent compound, anenzyme, or an enzyme co-factor. In another embodiment a set of primersis provided, e.g., primers suitable for use in a PCR, which can be usedto amplify a selected region of a TFM-2 and/or TFM-3 sequence, e.g., adomain, region, site or other sequence described herein. The primersshould be at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or morenucleotides in length. Such probes can be used as a part of a diagnostictest kit for identifying cells or tissue which misexpress a TFM-2 and/orTFM-3 polypeptide, such as by measuring a level of a TFM-2 and/orTFM-3-encoding nucleic acid in a sample of cells from a subject e.g.,detecting TFM-2 and/or TFM-3 mRNA levels or determining whether agenomic TFM-2 and/or TFM-3 gene has been mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion of aTFM-2 polypeptide” and/or a “biologically active portion of a TFM-3polypeptide” can be prepared by isolating a portion of the nucleotidesequence of SEQ ID NO:27, 29, 30, or 32, which encodes a polypeptidehaving a TFM-2 and/or TFM-3 biological activity (the biologicalactivities of the TFM-2 and/or TFM-3 polypeptides are described herein),expressing the encoded portion of the TFM-2 and/or TFM-3 polypeptide(e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion of the TFM-2 and/or TFM-3 polypeptide. In anexemplary embodiment, the nucleic acid molecule is at least 50-100,100-250, 250-500, 500-750, 750-1000, 1000-1250, 1250-1500, 1500-1750,1750-2000, 2000-2250, 2250-2500, 2500-2750, 2750-3000, 3000-3250,3250-3500 or more nucleotides in length and encodes a polypeptide havinga TFM-2 activity (as described herein). In another exemplary embodiment,the nucleic acid molecule is at least 50-100, 100-250, 250-500, 500-750,750-1000, 1000-1250, 1250-1500, 1500-1750, 1750-1850 or more nucleotidesin length and encodes a polypeptide having a TFM-3 activity (asdescribed herein).

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:27, 29, 30, or 32. Suchdifferences can be due to due to degeneracy of the genetic code, thusresulting in a nucleic acid which encodes the same TFM-2 and/or TFM-3polypeptides as those encoded by the nucleotide sequence shown in SEQ IDNO:27, 29, 30, or 32. In another embodiment, an isolated nucleic acidmolecule of the invention has a nucleotide sequence encoding apolypeptide having an amino acid sequence which differs by at least 1,but no greater than 5, 10, 20, 50 or 100 amino acid residues from theamino acid sequence shown in SEQ IfD NO:28 or 31. In yet anotherembodiment, the nucleic acid molecule encodes the amino acid sequence ofhuman TFM-2 and TFM-3. If an alignment is needed for this comparison,the sequences should be aligned for maximum homology.

Nucleic acid variants can be naturally occurring, such as allelicvariants (same locus), homologues (different locus), and orthologues(different organism) or can be non naturally occurring. Non-naturallyoccurring variants can be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions (as compared in the encoded product).

Allelic variants result, for example, from DNA sequence polymorphismswithin a population (e.g., the human population) that lead to changes inthe amino acid sequences of the TFM-2 and/or TFM-3 polypeptides. Suchgenetic polymorphism in the TFM-2 and/or TFM-3 genes may exist amongindividuals within a population due to natural allelic variation. Asused herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules which include an open reading frame encoding a TFM-2and/or TFM-3 polypeptide, preferably a mammalian TFM-2 and/or TFM-3polypeptide, and can further include non-coding regulatory sequences,and introns.

Accordingly, in one embodiment, the invention features isolated nucleicacid molecules which encode a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:28 or 31,wherein the nucleic acid molecule hybridizes to a complement of anucleic acid molecule comprising SEQ ID NO:27, 29, 30, or 32, forexample, under stringent hybridization conditions.

Allelic variants of human TFM-2 and/or TFM-3 include both functional andnon-functional TFM-2 and/or TFM-3 polypeptides. Functional allelicvariants are naturally occurring amino acid sequence variants of thehuman TFM-2 and/or TFM-3 polypeptide that have a TFM-2 and/or TFM-3activity, e.g., maintain the ability to bind a TFM-2 and/or TFM-3 ligandor substrate and/or modulate the import and export of molecules fromcells or across membranes, e.g., monocarboxylates and/ormonosaccharides. Functional allelic variants will typically contain onlyconservative substitution of one or more amino acids of SEQ ID NO:28 or31, or substitution, deletion or insertion of non-critical residues innon-critical regions of the polypeptide.

Non-functional allelic variants are naturally occurring amino acidsequence variants of the human TFM-2 and/or TFM-3 polypeptide that donot have a TFM-2 and/or TFM-3 activity, e.g., they do not have theability to transport molecules into and out of cells or acrossmembranes. Non-functional allelic variants will typically contain anon-conservative substitution, a deletion, or insertion or prematuretruncation of the amino acid sequence of SEQ ID NO:28 or 31, or asubstitution, insertion or deletion in critical residues or criticalregions.

The present invention further provides non-human orthologues of thehuman TFM-2 and/or TFM-3 polypeptide. Orthologues of human TFM-2 and/orTFM-3 polypeptides are polypeptides that are isolated from non-humanorganisms and possess the same TFM-2 and/or TFM-3 activity, e.g., ligandbinding and/or modulation of import and export of molecules from cellsor across membranes, e.g., monocarboxylates and/or monosaccharides, asthe human TFM-2 and/or TFM-3 polypeptide. Orthologues of the human TFM-2and/or TFM-3 polypeptide can readily be identified as comprising anamino acid sequence that is substantially identical to SEQ ID NO:28 or31.

Moreover, nucleic acid molecules encoding other TFM-2 and/or TFM-3family members and, thus, which have a nucleotide sequence which differsfrom the TFM-2 and/or TFM-3 sequences of SEQ ID NO:27, 29, 30, or 32,are intended to be within the scope of the invention. For example,another TFM-2 and/or TFM-3 cDNA can be identified based on thenucleotide sequence of human TFM-2 and/or TFM-3. Moreover, nucleic acidmolecules encoding TFM-2 and/or TFM-3 polypeptides from differentspecies, and which, thus, have a nucleotide sequence which differs fromthe TFM-2 and/or TFM-3 sequences of SEQ ID NO:27, 29, 30, or 32, areintended to be within the scope of the invention. For example, a mouseTFM-2 and/or TFM-3 cDNA can be identified based on the nucleotidesequence of a human TFM-2 and/or TFM-3.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the TFM-2 and/or TFM-3 cDNAs of the invention can beisolated based on their homology to the TFM-2 and/or TFM-3 nucleic acidsdisclosed herein using the cDNAs disclosed herein, or a portion thereof,as a hybridization probe according to standard hybridization techniquesunder stringent hybridization conditions. Nucleic acid moleculescorresponding to natural allelic variants and homologues of the TFM-2and/or TFM-3 cDNAs of the invention can further be isolated by mappingto the same chromosome or locus as the TFM-2 and/or TFM-3 gene.

Orthologues, homologues and allelic variants can be identified usingmethods known in the art (e.g., by hybridization to an isolated nucleicacid molecule of the present invention, for example, under stringenthybridization conditions). In one embodiment, an isolated nucleic acidmolecule of the invention is at least 15, 20, 25, 30 or more nucleotidesin length and hybridizes under stringent conditions to the nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:27, 29, 30, or32. In other embodiment, the nucleic acid is at least 100-150, 150-200,200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600,600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000,1000-1050, 1050-1100, 1100-1150, 1150-1200, 1200-1250, 1250-1300,1300-1350, 1350-1400, 1400-1450, 1450-1500, 1500-1550, 1550-1600,1600-1650, 1650-1700, 1700-1750, 1750-1800, 1800-1850, 1850-1900,1900-1950, 1950-2000, 2000-2500, 2500-3000, 3000-3500 or morenucleotides in length. In other embodiment, the nucleic acid is at least100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500,500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900,900-950, 950-1000, 1000-1050, 1050-1100, 1100-1150, 1150-1200,1200-1250, 1250-1300, 1300-1350, 1350-1400, 1400-1450, 1450-1500,1500-1550, 1550-1600, 1600-1650, 1650-1700, 1700-1750, 1750-1800,1800-1850 or more nucleotides in length.

In one embodiment, an isolated nucleic acid molecule of the inventioncomprises the nucleotide sequence shown in SEQ ID NO:33. The sequence ofSEQ ID NO:33 corresponds to the human 67118 cDNA. This cDNA comprisessequences encoding the human 67118 polypeptide (i.e., “the codingregion”, from nucleotides 94-3495) as well as 5′ untranslated sequences(nucleotides 1-83) and 3′ untranslated sequences (nucleotides3486-7745). Alternatively, the nucleic acid molecule can comprise onlythe coding region of SEQ ID NO:33 (e.g., nucleotides 84-3485,corresponding to SEQ ID NO:35). Accordingly, in another embodiment, theisolated nucleic acid molecule comprises SEQ ID NO:35 andnucleotides.1-84 and 3486-7745 of SEQ ID NO:33. In yet anotherembodiment, the nucleic acid molecule consists of the nucleotidesequence set forth as SEQ ID NO:33 or 35.

In another embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:36. Thesequence of SEQ ID NO:36 corresponds to the human 67067 cDNA. This cDNAcomprises sequences encoding the human 67067 polypeptide (i.e., “thecoding region”, from nucleotides 157-4920) as well as 5′ untranslatedsequences (nucleotides 1-156) and 3′ untranslated sequences (nucleotides4921-7205). Alternatively, the nucleic acid molecule can comprise onlythe coding region of SEQ ID NO:36 (e.g., nucleotides 157-4920,corresponding to SEQ ID NO:38). Accordingly, in another embodiment, theisolated nucleic acid molecule comprises SEQ ID NO:38 and nucleotides1-156 and 4921-7205 of SEQ ID NO:36. In yet another embodiment, thenucleic acid molecule consists of the nucleotide sequence set forth asSEQ ID NO:36 or 38.

In still another embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:39 or 41.This cDNA comprises sequences encoding the human 62092 protein (e.g.,the “coding region”, from nucleotides 357-845), as well as 5′untranslated sequence (nucleotides 1-356) and 3′ untranslated sequences(nucleotides 846-978) of SEQ ID NO:39. Alternatively, the nucleic acidmolecule can comprise only the coding region of SEQ ID NO:39 (e.g.,nucleotides 357-845, corresponding to SEQ ID NO:41). Accordingly, inanother embodiment, an isolated nucleic acid molecule of the inventioncomprises SEQ ID NO:41 and nucleotides 1-356 of SEQ ID NO:39. In yetanother embodiment, the isolated nucleic acid molecule comprises SEQ IDNO:41 and nucleotides 846-978 of SEQ ID NO:39. In yet anotherembodiment, the nucleic acid molecule consists of the nucleotidesequence set forth as SEQ ID NO:39 or 41.

In still another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO:33, 35, 36, 38, 39, or 41, or aportion of any of these nucleotide sequences. A nucleic acid moleculewhich is complementary to the nucleotide sequence shown in SEQ ID NO:33,35, 36, 38, 39, or 41, is one which is sufficiently complementary to thenucleotide sequence shown in SEQ ID NO:33, 35, 36, 38, 39, or 41, suchthat it can hybridize to the nucleotide sequence shown in SEQ ID NO:33,35, 36, 38, 39, or 41, thereby forming a stable duplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the present invention comprises a nucleotide sequence which is atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to the nucleotidesequence shown in SEQ ID NO:33, 35, 36, 38, 39, or 41 (e.g., to theentire length of the nucleotide sequence), or a portion of any of thesenucleotide sequences. In one embodiment, a nucleic acid molecule of thepresent invention comprises a nucleotide sequence which is at least (orno greater than) 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-1250,1250-1500, 1500-1750, 1750-2000, 2000-2250, 2250-2500, 2500-2750,2750-3000, 3000-3250, 3250-3500, 3500-3750, 3750-4000, 4000-4250,4250-4500, 4500-4750, 4750-5000, 5000-5250, 5250-5500, 5500-5750,5750-6000, 6000-6250, 6250-6500, 6500-6750, 6750-7000, 7000-7250,7250-7500 or more nucleotides in length and hybridizes under stringenthybridization conditions to a complement of a nucleic acid molecule ofSEQ ID NO:33, 35, 36, 38, 39, 41.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO:33, 35, 36, 38, 39,41, for example, a fragment which can be used as a probe or primer or afragment encoding a portion of a 67118, 67067, and/or 62092 polypeptide,e.g., a biologically active portion of a 67118, 67067, and/or 62092polypeptide. The nucleotide sequence determined from the cloning of the67118, 67067, and/or 62092 gene allows for the generation of probes andprimers designed for use in identifying and/or cloning other 67118,67067, and/or 62092 family members, as well as 67118, 67067, and/or62092 homologues from other species. The probe/primer typicallycomprises substantially purified oligonucleotide. The probe/primer(e.g., oligonucleotide) typically comprises a region of nucleotidesequence that hybridizes under stringent conditions to at least about 12or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45,50, 55, 60, 65, 75, 80, 85, 90, 95, or 100 or more consecutivenucleotides of a sense sequence of SEQ ID NO: 33, 35, 36, 38, 39, 41, ofan anti-sense sequence of SEQ ID NO:33, 35, 36, 38, 39, 41, or of anaturally occurring allelic variant or mutant of SEQ ID NO:33, 35, 36,38, 39, 41.

Exemplary probes or primers are at least 12, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75 or more nucleotides in length and/or compriseconsecutive nucleotides of an isolated nucleic acid molecule describedherein. Probes based on the 67118, 67067, and/or 62092 nucleotidesequences can be used to detect (e.g., specifically detect) transcriptsor genomic sequences encoding the same or homologous polypeptides. Inpreferred embodiments, the probe further comprises a label groupattached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. In anotherembodiment a set of primers is provided, e.g., primers suitable for usein a PCR, which can be used to amplify a selected region of a 67118,67067, and/or 62092 sequence, e.g., a domain, region, site or othersequence described herein. The primers should be at least 5, 10, 20, 30,40, 50, 60, 70, 80, 90, 100 or more nucleotides in length. Such probescan be used as a part of a diagnostic test kit for identifying cells ortissue which misexpress a 67118, 67067, and/or 62092 polypeptide, suchas by measuring a level of a 67118, 67067, and/or 62092-encoding nucleicacid in a sample of cells from a subject e.g., detecting 67118, 67067,and/or 62092 mRNA levels or determining whether a genomic 67118, 67067,and/or 62092 gene has been mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion of a67118 polypeptide,” a “biologically active portion of a 67067polypeptide,” or a “biologically active portion of a 62092 polypeptide,”can be prepared by isolating a portion of the nucleotide sequence of SEQID NO:33, 35, 36, 38, 39, 41, which encodes a polypeptide having a67118, 67067, and/or 62092 biological activity (the biologicalactivities of the 67118, 67067, and/or 62092 polypeptides are describedherein), expressing the encoded portion of the 67118, 67067, and/or62092 polypeptide (e.g., by recombinant expression in vitro) andassessing the activity of the encoded portion of the 67118, 67067,and/or 62092 polypeptide. In an exemplary embodiment, the nucleic acidmolecule is at least 50-100, 100-250, 250-500, 500-750, 750-1000,1000-1250, 1250-1500, 1500-1750, 1750-2000, 2000-2250, 2250-2500,2500-2750, 2750-3000, 3000-3250, 3250-3500, 3500-3750, 3750-4000,4000-4250, 4250-4500, 4500-4750, 4750-5000, 5000-5250, 5250-5500,5500-5750, 5750-6000, 6000-6250, 6250-6500, 6500-6750, 6750-7000,7000-7250, 7250-7500 or more nucleotides in length and encodes apolypeptide having a 67118, 67067, and/or 62092 activity (as describedherein).

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:33, 35, 36, 38, 39, or41. Such differences can be due to due to degeneracy of the geneticcode, thus resulting in a nucleic acid which encodes the same 67118,67067, and/or 62092 polypeptides as those encoded by the nucleotidesequence shown in SEQ ID NO:33, 35, 36, 38, 39, or 41. In anotherembodiment, an isolated nucleic acid molecule of the invention has anucleotide sequence encoding a polypeptide having an amino acid sequencewhich differs by at least 1, but no greater than 5, 10, 20, 50 or 100amino acid residues from the amino acid sequence shown in SEQ ID NO:34,37, or 40. In yet another embodiment, the nucleic acid molecule encodesthe amino acid sequence of human 67118, 67067, and/or 62092. If analignment is needed for this comparison, the sequences should be alignedfor maximum homology.

Nucleic acid variants can be naturally occurring, such as allelicvariants (same locus), homologues (different locus), and orthologues(different organism) or can be non naturally occurring. Non-naturallyoccurring variants can be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions (as compared in the encoded product).

Allelic variants result, for example, from DNA sequence polymorphismswithin a population (e.g., the human population) that lead to changes inthe amino acid sequences of the 67118, 67067, and/or 62092 polypeptides.Such genetic polymorphism in the 67118, 67067, and/or 62092 genes mayexist among individuals within a population due to natural allelicvariation. As used herein, the terms “gene” and “recombinant gene” referto nucleic acid molecules which include an open reading frame encoding a67118, 67067, and/or 62092 polypeptide, preferably a mammalian 67118,67067, and/or 62092 polypeptide, and can further include non-codingregulatory sequences, and introns.

Accordingly, in one embodiment, the invention features isolated nucleicacid molecules which encode a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:34, 37, or40, wherein the nucleic acid molecule hybridizes to a complement of anucleic acid molecule comprising SEQ ID NO:33, 35, 36, 38, 39, or 41,for example, under stringent hybridization conditions.

Allelic variants of human 67118, 67067, and/or 62092 include bothfunctional and non-functional 67118, 67067, and/or 62092 polypeptides.Functional allelic variants are naturally occurring amino acid sequencevariants of the human 67118 or 67067 polypeptide that have a 67118 or67067 activity, e.g., bind or interact with a 67118 or 67067 substrateor target molecule, transport a 67118 or 67067 substrate or targetmolecule (e.g., a phospholipid) across a cellular membrane, hydrolyzeATP, be phosphorylated or dephosphorylated, adopt an E1 conformation oran E2 conformation, and/or modulate cellular signaling, growth,proliferation, differentiation, absorption, or secretion. Functionalallelic variants will typically contain only conservative substitutionof one or more amino acids of SEQ ID NO:34 or 37, or substitution,deletion or insertion of non-critical residues in non-critical regionsof the polypeptide. Functional allelic variants are naturally occurringamino acid sequence variants of the 62092 protein that maintain theability to, e.g., bind or interact with a 62092 substrate or targetmolecule and/or modulate cellular signaling and/or gene transcription.Functional allelic variants will typically contain only conservativesubstitution of one or more amino acids of SEQ ID NO:40, orsubstitution, deletion or insertion of non-critical residues innon-critical regions of the protein.

Non-functional allelic variants are naturally occurring amino acidsequence variants of the human 67118 or 67067 polypeptide that do nothave a 67118 or 67067 activity, e.g., that do not have the ability to,e.g., bind or interact with a 67118 or 67067 substrate or targetmolecule, transport a 67118 or 67067 substrate or target molecule (e.g.,a phospholipid) across a cellular membrane, hydrolyze ATP, bephosphorylated or dephosphorylated, adopt an E1 conformation or an E2conformation, and/or modulate cellular signaling, growth, proliferation,differentiation, absorption, or secretion. Non-functional allelicvariants will typically contain a non-conservative substitution, adeletion, or insertion or premature truncation of the amino acidsequence of SEQ ID NO:34 or 37, or a substitution, insertion or deletionin critical residues or critical regions. Moreover, non-functionalallelic variants are naturally occurring amino acid sequence variants ofthe 62092 protein, e.g., human 62092, that do not have the ability to,e.g., bind or interact with a 62092 substrate or target molecule and/ormodulate cellular signaling and/or gene transcription. Non-functionalallelic variants will typically contain a non-conservative substitution,a deletion, or insertion, or premature truncation of the amino acidsequence of SEQ ID NO:40, or a substitution, insertion, or deletion incritical residues or critical regions of the protein.

The present invention further provides non-human orthologues of thehuman 67118, 67067, and/or 62092 polypeptides. Orthologues of human67118 or 67067 polypeptides are polypeptides that are isolated fromnon-human organisms and possess the same 67118 or 67067 substrate ortarget molecule binding mechanisms, phospholipid transporting activity,ATPase activity, and/or modulation of cellular signaling mechanisms ofthe human PLTR proteins as the human 67118 or 67067 polypeptides.Orthologues of the human 67118 or 67067 polypeptides can readily beidentified as comprising an amino acid sequence that is substantiallyidentical to SEQ ID NO:34 or 37. Orthologues of the human 62092 proteinare proteins that are isolated from non-human organisms and possess thesame 62092 substrate or target molecule binding mechanisms and/orability to modulate cellular signaling and/or gene transcription of thehuman 62092 protein. Orthologues of the human 62092 protein can readilybe identified as comprising an amino acid sequence that is substantiallyhomologous to SEQ ID NO:40.

Moreover, nucleic acid molecules encoding other 67118, 67067, and/or62092 family members and, thus, which have a nucleotide sequence whichdiffers from the 67118, 67067, and/or 62092 sequences of SEQ ID NO:33,35, 36, 38, 39, or 41, are intended to be within the scope of theinvention. For example, another 67118, 67067, and/or 62092 cDNA can beidentified based on the nucleotide sequence of human 67118, 67067,and/or 62092. Moreover, nucleic acid molecules encoding 67118, 67067,and/or 62092 polypeptides from different species, and which, thus, havea nucleotide sequence which differs from the 67118, 67067, and/or 62092sequences of SEQ ID NO:33, 35, 36, 38, 39, or 41, are intended to bewithin the scope of the invention. For example, a mouse 67118, 67067,and/or 62092 cDNA can be identified based on the nucleotide sequence ofa human 67118, 67067, and/or 62092.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the 67118, 67067, and/or 62092 cDNAs of the invention canbe isolated based on their homology to the 67118, 67067, and/or 62092nucleic acids disclosed herein using the cDNAs disclosed herein, or aportion thereof, as a hybridization probe according to standardhybridization techniques under stringent hybridization conditions.Nucleic acid molecules corresponding to natural allelic variants andhomologues of the 67118, 67067, and/or 62092 cDNAs of the invention canfurther be isolated by mapping to the same chromosome or locus as the67118, 67067, and/or 62092 gene.

Orthologues, homologues and allelic variants can be identified usingmethods known in the art (e.g., by hybridization to an isolated nucleicacid molecule of the present invention, for example, under stringenthybridization conditions). In one embodiment, an isolated nucleic acidmolecule of the invention is at least 15, 20, 25, 30 or more nucleotidesin length and hybridizes under stringent conditions to the nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:33, 35, 36, 38,39, or 41. In other embodiment, the nucleic acid is at least 50-100,100-250, 250-500, 500-750, 750-1000, 1000-1250, 1250-1500, 1500-1750,1750-2000, 2000-2250, 2250-2500, 2500-2750, 2750-3000, 3000-3250,3250-3500, 3500-3750, 3750-4000, 4000-4250, 4250-4500, 4500-4750,4750-5000, 5000-5250, 5250-5500, 5500-5750, 5750-6000, 6000-6250,6250-6500, 6500-6750, 6750-7000, 7000-7250, 7250-7500 or morenucleotides in length.

In one embodiment, an isolated nucleic acid molecule of the inventioncomprises the nucleotide sequence shown in SEQ ID NO:51 or 53. This cDNAmay comprise sequences encoding the human HAAT protein (e.g., the“coding region”, from nucleotides 69-1526), as well as 5′ untranslatedsequence (nucleotides 1-68) and 3′ untranslated sequences (nucleotides1527-2397) of SEQ ID NO:51. Alternatively, the nucleic acid molecule cancomprise only the coding region of SEQ ID NO:51 (e.g., nucleotides69-1526, corresponding to SEQ ID NO:53). Accordingly, in anotherembodiment, an isolated nucleic acid molecule of the invention comprisesSEQ ID NO:53 and nucleotides 1-68 of SEQ ID NO:51. In yet anotherembodiment, the isolated nucleic acid molecule comprises SEQ ID NO:53and nucleotides 1527-2397 of SEQ ID NO:51. In yet another embodiment,the nucleic acid molecule consists of the nucleotide sequence set forthas SEQ ID NO:51 or 53.

In still another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO:51 or 53, or a portion of any ofthese nucleotide sequences. A nucleic acid molecule which iscomplementary to the nucleotide sequence shown in SEQ ID NO:51 or 53, isone which is sufficiently complementary to the nucleotide sequence shownin SEQ ID NO:51 or 53, such that it can hybridize to the nucleotidesequence shown in SEQ ID NO:51 or 53, thereby forming a stable duplex.

In still another embodiment, an isolated nucleic acid molecule of thepresent invention comprises a nucleotide sequence which is at leastabout 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical tothe nucleotide sequence shown in SEQ ID NO:51 or 53 (e.g., to the entirelength of the nucleotide sequence), or a portion or complement of any ofthese nucleotide sequences. In one embodiment, a nucleic acid moleculeof the present invention comprises a nucleotide sequence which is atleast (or no greater than) 50-100, 100-250, 250-500, 500-750, 750-1000,1000-1250, 1250-1500, 1500-1750, 1750-2000, 2000-2250, 2250-2500,2500-2750, 2750-3000 or more nucleotides in length and hybridizes understringent hybridization conditions to a complement of a nucleic acidmolecule of SEQ ID NO:51 or 53.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO:51 or 53, for example,a fragment which can be used as a probe or primer or a fragment encodinga portion of a HAAT protein, e.g., a biologically active portion of aHAAT protein. The nucleotide sequence determined from the cloning of theHAAT gene allows for the generation of probes and primers designed foruse in identifying and/or cloning other HAAT family members, as well asHAAT homologues from other species. The probe/primer (e.g.,oligonucleotide) typically comprises substantially purifiedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12 or 15, preferably about 20 or 25, more preferably about30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sensesequence of SEQ ID NO:51 or 53, of an anti-sense sequence of SEQ IDNO:51 or 53, or of a naturally occurring allelic variant or mutant ofSEQ ID NO:51 or 53. In another embodiment, a fragment comprises at least8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350,400, 450, 475, 500, 550, 575, 600, 650 or more nucleic acids (e.g.,contiguous or consecutive nucleotides) of the nucleotide sequence of SEQID NO:51 or 53, or of a naturally occurring allelic variant or mutant ofSEQ ID NO:51 or 53.

Exemplary probes or primers are at least (or no greater than) 12 or 15,20 or 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more nucleotides inlength and/or comprise consecutive nucleotides of an isolated nucleicacid molecule described herein. Also included within the scope of thepresent invention are probes or primers comprising contiguous orconsecutive nucleotides of an isolated nucleic acid molecule describedherein, but for the difference of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 baseswithin the probe or primer sequence. Probes based on the HAAT nucleotidesequences can be used to detect (e.g., specifically detect) transcriptsor genomic sequences encoding the same or homologous proteins. Inpreferred embodiments, the probe further comprises a label groupattached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. In anotherembodiment a set of primers is provided, e.g., primers suitable for usein a PCR, which can be used to amplify a selected region of a HAATsequence, e.g., a domain, region, site or other sequence describedherein. The primers should be at least 5, 10, or 50 base pairs in lengthand less than 100, or less than 200, base pairs in length. The primersshould be identical, or differ by no greater than 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 bases when compared to a sequence disclosed herein or to thesequence of a naturally occurring variant. Such probes can be used as apart of a diagnostic test kit for identifying cells or tissue whichmisexpress a HAAT protein, such as by measuring a level of aHAAT-encoding nucleic acid in a sample of cells from a subject, e.g.,detecting HAAT mRNA levels or determining whether a genomic HAAT genehas been mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion of aHAAT protein” can be prepared by isolating a portion of the nucleotidesequence of SEQ ID NO:51 or 53, which encodes a polypeptide having aHAAT biological activity (the biological activities of the HAAT proteinsare described herein), expressing the encoded portion of the HAATprotein (e.g., by recombinant expression in vitro) and assessing theactivity of the encoded portion of the HAAT protein. In an exemplaryembodiment, the nucleic acid molecule is at least 50-100, 100-250,250-500, 500-700, 750-1000, 1000-1250, 1250-1500, 1500-1750, 1750-2000,2000-2250, 2250-2400 or more nucleotides in length and encodes a proteinhaving a HAAT activity (as described herein).

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:51 or 53, due todegeneracy of the genetic code and thus encode the same HAAT proteins asthose encoded by the nucleotide sequence shown in SEQ ID NO:51 or 53. Inanother embodiment, an isolated nucleic acid molecule of the inventionhas a nucleotide sequence encoding a protein having an amino acidsequence which differs by at least 1, but no greater than 5, 10, 20, 50or 100 amino acid residues from the amino acid sequence shown in SEQ IDNO:52. In yet another embodiment, the nucleic acid molecule encodes theamino acid sequence of human HAAT. If an alignment is needed for thiscomparison, the sequences should be aligned for maximum homology.

Nucleic acid variants can be naturally occurring, such as allelicvariants (same locus), homologues (different locus), and orthologues(different organism) or can be non naturally occurring. Non-naturallyoccurring variants can be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions (as compared in the encoded product).

Allelic variants result, for example, from DNA sequence polymorphismswithin a population (e.g., the human population) that lead to changes inthe amino acid sequences of the HAAT proteins. Such genetic polymorphismin the HAAT genes may exist among individuals within a population due tonatural allelic variation. As used herein, the terms “gene” and“recombinant gene” refer to nucleic acid molecules which include an openreading frame encoding a HAAT protein, preferably a mammalian HAATprotein, and can further include non-coding regulatory sequences, andintrons.

Accordingly, in one embodiment, the invention features isolated nucleicacid molecules which encode a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:52, whereinthe nucleic acid molecule hybridizes to a complement of a nucleic acidmolecule comprising SEQ ID NO:51 or 53, for example, under stringenthybridization conditions.

Allelic variants of HAAT, e.g., human HAAT, include both functional andnon-functional HAAT proteins. Functional allelic variants are naturallyoccurring amino acid sequence variants of the HAAT protein that maintainthe ability to, e.g., bind or interact with a HAAT substrate or targetmolecule, transport a HAAT substrate or target molecule (e.g., an aminoacid) across a cellular membrane and/or modulate protein synthesis,hormone metabolism, nerve transmission, cellular activation, regulationof cell growth, production of metabolic energy, synthesis of purines andpyrimidines, nitrogen metabolism, and/or biosynthesis of urea.Functional allelic variants will typically contain only conservativesubstitution of one or more amino acids of SEQ ID NO:52, orsubstitution, deletion or insertion of non-critical residues innon-critical regions of the protein.

Non-functional allelic variants are naturally occurring amino acidsequence variants of the HAAT protein, e.g., human HAAT, that do nothave the ability to, e.g., bind or interact with a HAAT substrate ortarget molecule, transport a HAAT substrate or target molecule (e.g., anamino acid) across a cellular membrane and/or modulate proteinsynthesis, hormone metabolism, nerve transmission, cellular activation,regulation of cell growth, production of metabolic energy, synthesis ofpurines and pyrimidines, nitrogen metabolism, and/or biosynthesis ofurea. Non-functional allelic variants will typically contain anon-conservative substitution, a deletion, or insertion, or prematuretruncation of the amino acid sequence of SEQ ID NO:52, or asubstitution, insertion, or deletion in critical residues or criticalregions of the protein.

The present invention further provides non-human orthologues (e.g.,non-human orthologues of the human HAAT protein). Orthologues of thehuman HAAT protein are proteins that are isolated from non-humanorganisms and possess the same HAAT substrate or target molecule bindingmechanisms, amino acid transporting activity and/or modulation ofnitrogen metabolism mechanisms of the human HAAT proteins. Orthologuesof the human HAAT protein can readily be identified as comprising anamino acid sequence that is substantially homologous to SEQ ID NO:52.

Moreover, nucleic acid molecules encoding other HAAT family members and,thus, which have a nucleotide sequence which differs from the HAATsequences of SEQ ID NO:51 or 53, are intended to be within the scope ofthe invention. For example, another HAAT cDNA can be identified based onthe nucleotide sequence of human HAAT. Moreover, nucleic acid moleculesencoding HAAT proteins from different species, and which, thus, have anucleotide sequence which differs from the HAAT sequences of SEQ IDNO:51 or 53, are intended to be within the scope of the invention. Forexample, a mouse or monkey HAAT cDNA can be identified based on thenucleotide sequence of a human HAAT.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the HAAT cDNAs of the invention can be isolated based ontheir homology to the HAAT nucleic acids disclosed herein using thecDNAs disclosed herein, or a portion thereof, as a hybridization probeaccording to standard hybridization techniques under stringenthybridization conditions. Nucleic acid molecules corresponding tonatural allelic variants and homologues of the HAAT cDNAs of theinvention can further be isolated by mapping to the same chromosome orlocus as the HAAT gene.

Orthologues, homologues and allelic variants can be identified usingmethods known in the art (e.g., by hybridization to an isolated nucleicacid molecule of the present invention, for example, under stringenthybridization conditions). In one embodiment, an isolated nucleic acidmolecule of the invention is at least 15, 20, 25, 30 or more nucleotidesin length and hybridizes under stringent conditions to the nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:51 or 53. Inother embodiment, the nucleic acid is at least 50-100, 100-250, 250-500,500-700, 750-1000, 1000-1250, 1250-1500, 1500-1750, 1750-2000,2000-2250, 2250-2400 or more nucleotides in length (e.g., 2397nucleotides in length).

In one embodiment, an isolated nucleic acid molecule of the inventioncomprises the nucleotide sequence shown in SEQ ID NO:54. The sequence ofSEQ ID NO:54 corresponds to the human HST-4 cDNA. This cDNA comprisessequences encoding the human HST-4 polypeptide (i.e., “the codingregion”, from nucleotides 137-1450) as well as 5′ untranslated sequences(nucleotides 1-136) and 3′ untranslated sequences (nucleotides1451-2565). Alternatively, the nucleic acid molecule can comprise onlythe coding region of SEQ ID NO:54 (e.g., nucleotides 137-1450,corresponding to SEQ ID NO:56). Accordingly, in another embodiment, theisolated nucleic acid molecule comprises SEQ ID NO:56 and nucleotides1-136 and 1451-2565 of SEQ ID NO:54. In yet another embodiment, thenucleic acid molecule consists of the nucleotide sequence set forth asSEQ ID NO:54 or SEQ ID NO:56.

In another embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:57. Thesequence of SEQ ID NO:57 corresponds to the human HST-5 cDNA. This cDNAcomprises sequences encoding the human HST-5 polypeptide (i.e., “thecoding region”, from nucleotides 137-1444) as well as 5′ untranslatedsequences (nucleotides 1-136) and 3′ untranslated sequences (nucleotides1445-2558). Alternatively, the nucleic acid molecule can comprise onlythe coding region of SEQ ID NO:57 (e.g., nucleotides 137-1444,corresponding to SEQ ID NO:59). Accordingly, in another embodiment, theisolated nucleic acid molecule comprises SEQ ID NO:59 and nucleotides1-136 and 1445-2558 of SEQ ID NO:57. In yet another embodiment, thenucleic acid molecule consists of the nucleotide sequence set forth asSEQ ID NO:57 or SEQ ID NO:59.

In still another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO:54, 56, 57, or 59, or a portionof any of these nucleotide sequences. A nucleic acid molecule which iscomplementary to the nucleotide sequence shown in SEQ ID NO:54, 56, 57,or 59, is one which is sufficiently complementary to the nucleotidesequence shown in SEQ ID NO:54, 56, 57, or 59, such that it canhybridize to the nucleotide sequence shown in SEQ ID NO:54, 56, 57, or59, thereby forming a stable duplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the present invention comprises a nucleotide sequence which is atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to the nucleotidesequence shown in SEQ ID NO:54, 56, 57, or 59 (e.g., to the entirelength of the nucleotide sequence), or a portion of any of thesenucleotide sequences. In one embodiment, a nucleic acid molecule of thepresent invention comprises a nucleotide sequence which is at least 10,20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500,1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500 or morenucleotides in length and hybridizes under stringent hybridizationconditions to a complement of a nucleic acid molecule of SEQ ID NO:54,56, 57, or 59. In another embodiment, a nucleic acid molecule of thepresent invention comprises a nucleotide sequence which is at least 10,20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500,1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500 or morenucleotides in length and hybridizes under stringent hybridizationconditions to a complement of a nucleic acid molecule of SEQ ID NO:54,56, 57, or 59.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO:54, 56, 57, or 59, forexample, a fragment which can be used as a probe or primer or a fragmentencoding a portion of an HST-4 and/or HST-5 polypeptide, e.g., abiologically active portion of an HST-4 and/or HST-5 polypeptide. Thenucleotide sequence determined from the cloning of the HST-4 and/orHST-5 gene allows for the generation of probes and primers designed foruse in identifying and/or cloning other HST-4 and/or HST-5 familymembers, as well as HST-4 and/or HST-5 homologues from other species.The probe/primer typically comprises substantially purifiedoligonucleotide. The probe/primer (e.g., oligonucleotide) typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12 or 15, preferably about 20 or25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, 75, 80, 85,90, 95, or 100 or more consecutive nucleotides of a sense sequence ofSEQ ID NO:54, 56, 57, or 59, of an anti-sense sequence of SEQ ID NO:54,56, 57, or 59, or of a naturally occurring allelic variant or mutant ofSEQ ID NO:54, 56, 57, or 59.

Exemplary probes or primers are at least 12, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75 or more nucleotides in length and/or compriseconsecutive nucleotides of an isolated nucleic acid molecule describedherein. Probes based on the HST-4 and/or HST-5 nucleotide sequences canbe used to detect (e.g., specifically detect) transcripts or genomicsequences encoding the same or homologous polypeptides. In preferredembodiments, the probe further comprises a label group attached thereto,e.g., the label group can be a radioisotope, a fluorescent compound, anenzyme, or an enzyme co-factor. In another embodiment a set of primersis provided, e.g., primers suitable for use in a PCR, which can be usedto amplify a selected region of an HST-4 and/or HST-5 sequence, e.g., adomain, region, site or other sequence described herein. The primersshould be at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or morenucleotides in length. Such probes can be used as a part of a diagnostictest kit for identifying cells or tissue which misexpress an HST-4and/or HST-5 polypeptide, such as by measuring a level of an HST-4and/or HST-5-encoding nucleic acid in a sample of cells from a subjecte.g., detecting HST-4 and/or HST-5 mRNA levels or determining whether agenomic HST-4 and/or HST-5 gene has been mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion of anHST-4 polypeptide” or a “biologically active portion of an HST-5polypeptide” can be prepared by isolating a portion of the nucleotidesequence of SEQ ID NO:54, 56, 57, or 59, which encodes a polypeptidehaving an HST-4 and/or HST-5 biological activity (the biologicalactivities of the HST-4 and/or HST-5 polypeptides are described herein),expressing the encoded portion of the HST-4 and/or HST-5 polypeptide(e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion of the HST-4 and/or HST-5 polypeptide. In anexemplary embodiment, the nucleic acid molecule is at least 10, 20, 30,40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500 or more nucleotides inlength and encodes a polypeptide having an HST-4 activity (as describedherein). In another exemplary embodiment, the nucleic acid molecule isat least 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400,2500 or more nucleotides in length and encodes a polypeptide having anHST-5 activity (as described herein).

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:54, 56, 57, or 59. Suchdifferences can be due to due to degeneracy of the genetic code, thusresulting in a nucleic acid which encodes the same HST-4 and/or HST-5polypeptides as those encoded by the nucleotide sequence shown in SEQ IDNO:54, 56, 57, or 59. In another embodiment, an isolated nucleic acidmolecule of the invention has a nucleotide sequence encoding apolypeptide having an amino acid sequence which differs by at least 1,but no greater than 5, 10, 20, 50 or 100 amino acid residues from theamino acid sequence shown in SEQ ID NO:55 or 58. In yet anotherembodiment, the nucleic acid molecule encodes the amino acid sequence ofhuman HST-4 and/or HST-5. If an alignment is needed for this comparison,the sequences should be aligned for maximum homology.

Nucleic acid variants can be naturally occurring, such as allelicvariants (same locus), homologues (different locus), and orthologues(different organism) or can be non naturally occurring. Non-naturallyoccurring variants can be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions (as compared in the encoded product).

Allelic variants result, for example, from DNA sequence polymorphismswithin a population (e.g., the human population) that lead to changes inthe amino acid sequences of the HST-4 and/or HST-5 polypeptides. Suchgenetic polymorphism in the HST-4 and/or HST-5 genes may exist amongindividuals within a population due to natural allelic variation. Asused herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules which include an open reading frame encoding an HST-4and/or HST-5 polypeptide, preferably a mammalian HST-4 and/or HST-5polypeptide, and can further include non-coding regulatory sequences,and introns.

Accordingly, in one embodiment, the invention features isolated nucleicacid molecules which encode a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:55 or 58,wherein the nucleic acid molecule hybridizes to a complement of anucleic acid molecule comprising SEQ ID NO:54, 56, 57, or 59 forexample, under stringent hybridization conditions.

Allelic variants of human HST-4 and/or HST-5 include both functional andnon-functional HST-4 and/or HST-5 polypeptides. Functional allelicvariants are naturally occurring amino acid sequence variants of thehuman HST-4 and/or HST-5 polypeptide that have an HST-4 and/or HST-5activity, e.g., maintain the ability to bind an HST-4 and/or HST-5ligand or substrate and/or modulate sugar transport, or sugarhomeostasis. Functional allelic variants will typically contain onlyconservative substitution of one or more amino acids of SEQ ID NO:55 or58, or substitution, deletion or insertion of non-critical residues innon-critical regions of the polypeptide.

Non-functional allelic variants are naturally occurring amino acidsequence variants of the human HST-4 and/or HST-5 polypeptide that donot have an HST-4 and/or HST-5 activity, e.g., they do not have theability to transport sugars into and out of cells or to modulate sugarhomeostasis. Non-functional allelic variants will typically contain anon-conservative substitution, a deletion, or insertion or prematuretruncation of the amino acid sequence of SEQ ID NO:55 or 58, or asubstitution, insertion or deletion in critical residues or criticalregions.

The present invention further provides non-human orthologues of thehuman HST-4 and/or HST-5 polypeptide. Orthologues of human HST-4 and/orHST-5 polypeptides are polypeptides that are isolated from non-humanorganisms and possess the same HST-4 and/or HST-5 activity, e.g., ligandbinding and/or modulation of sugar transport mechanisms, as the humanHST-4 and/or HST-5 polypeptide. Orthologues of the human HST-4 and/orHST-5 polypeptide can readily be identified as comprising an amino acidsequence that is substantially identical to SEQ ID NO:55 or 58.

Moreover, nucleic acid molecules encoding other HST-4 and/or HST-5family members and, thus, which have a nucleotide sequence which differsfrom the HST-4 and/or HST-5 sequences of SEQ ID NO:54, 56, 57, or 59,are intended to be within the scope of the invention. For example,another HST-4 and/or HST-5 cDNA can be identified based on thenucleotide sequence of human HST-4 and/or HST-5. Moreover, nucleic acidmolecules encoding HST-4 and/or HST-5 polypeptides from differentspecies, and which, thus, have a nucleotide sequence which differs fromthe HST-4 and/or HST-5 sequences of SEQ ID NO:54, 56, 57, or 59, areintended to be within the scope of the invention. For example, a mouseHST-4 and/or HST-5 cDNA can be identified based on the nucleotidesequence of a human HST-4 and/or HST-5.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the HST-4 and/or HST-5 cDNAs of the invention can beisolated based on their homology to the HST-4 and/or HST-5 nucleic acidsdisclosed herein using the cDNAs disclosed herein, or a portion thereof,as a hybridization probe according to standard hybridization techniquesunder stringent hybridization conditions. Nucleic acid moleculescorresponding to natural allelic variants and homologues of the HST-4and/or HST-5 cDNAs of the invention can further be isolated by mappingto the same chromosome or locus as the HST-4 and/or HST-5 gene.

Orthologues, homologues and allelic variants can be identified usingmethods known in the art (e.g., by hybridization to an isolated nucleicacid molecule of the present invention, for example, under stringenthybridization conditions). In one embodiment, an isolated nucleic acidmolecule of the invention is at least 15, 20, 25, 30 or more nucleotidesin length and hybridizes under stringent conditions to the nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:54, 56, 57, or59. In other embodiment, the nucleic acid is at least 10, 20, 30, 40,50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500 or more nucleotides inlength.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences that are significantly identical orhomologous to each other remain hybridized to each other. Preferably,the conditions are such that sequences at least about 70%, morepreferably at least about 80%, even more preferably at least about 85%or 90% identical to each other remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, Ausubel et al., eds.,John Wiley & Sons, Inc. (1995), sections 2, 4 and 6. Additionalstringent conditions can be found in Molecular Cloning: A LaboratoryManual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor,N.Y. (1989), chapters 7, 9 and 11. A preferred, non-limiting example ofstringent hybridization conditions includes hybridization in 4× sodiumchloride/sodium citrate (SSC), at about 65-70° C. (or hybridization in4×SSC plus 50% formamide at about 42-50° C.) followed by one or morewashes in 1×SSC, at about 65-70° C. A preferred, non-limiting example ofhighly stringent hybridization conditions includes hybridization in1×SSC, at about 65-70° C. (or hybridization in 1×SSC plus 50% formamideat about 42-50° C.) followed by one or more washes in 0.3×SSC, at about65-70° C. A preferred, non-limiting example of reduced stringencyhybridization conditions includes hybridization in 4×SSC, at about50-60° C. (or alternatively hybridization in 6×SSC plus 50% formamide atabout 40-45° C.) followed by one or more washes in 2×SSC, at about50-60° C. Ranges intermediate to the above-recited values, e.g., at65-70° C. or at 42-50° C. are also intended to be encompassed by thepresent invention. SSPE (1×SSPE is 0.15M NaCl, 10 mM NaH₂PO₄, and 1.25mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.15M NaCl and 15mM sodium citrate) in the hybridization and wash buffers; washes areperformed for 15 minutes each after hybridization is complete. Thehybridization temperature for hybrids anticipated to be less than 50base pairs in length should be 5-10° C. less than the meltingtemperature (T_(m)) of the hybrid, where T_(m) is determined accordingto the following equations. For hybrids less than 18 base pairs inlength, T_(m)(° C.)=2(# of A+T bases)+4(# of G+C bases). For hybridsbetween 18 and 49 base pairs in length, T_(m)(°C.)=81.5+16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N), where N is the number ofbases in the hybrid, and [Na⁺] is the concentration of sodium ions inthe hybridization buffer ([Na⁺] for 1×SSC=0.165 M). It will also berecognized by the skilled practitioner that additional reagents may beadded to hybridization and/or wash buffers to decrease non-specifichybridization of nucleic acid molecules to membranes, for example,nitrocellulose or nylon membranes, including but not limited to blockingagents (e.g., BSA or salmon or herring sperm carrier DNA), detergents(e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like.When using nylon membranes, in particular, an additional preferred,non-limiting example of stringent hybridization conditions ishybridization in 0.25-0.5M NaH₂PO₄, 7% SDS at about 65° C., followed byone or more washes at 0.02M NaH₂PO₄, 1% SDS at 65° C., see e.g., Churchand Gilbert (1984) Proc. Natl. Acad. Sci. USA 81:1991-1995, (oralternatively 0.2×SSC, 1% SDS).

Preferably, an isolated nucleic acid molecule of the invention thathybridizes under stringent conditions to the sequence of SEQ ID NO:1, 3,4, 6, 7, 9, 12, 14, 15, 17, 19, 21, 27, 29, 30, 32, 33, 35, 36, 38, 39,41, 51, 53, 54, 56, 57, or 59, and corresponds to a naturally-occurringnucleic acid molecule. As used herein, a “naturally-occurring” nucleicacid molecule refers to an RNA or DNA molecule having a nucleotidesequence that occurs in nature (e.g., encodes a natural polypeptide).

In addition to naturally-occurring allelic variants of the MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or the HST-5 sequences that may exist in the population, the skilledartisan will further appreciate that changes can be introduced bymutation into the nucleotide sequences of SEQ ID NO:1, 3, 4, 6, 7, 9,12, 14, 15, 17, 19, 21, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 51, 53,54, 56, 57, or 59, thereby leading to changes in the amino acid sequenceof the encoded MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 polypeptides, without alteringthe functional ability of the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or the HST-5 polypeptides.For example, nucleotide substitutions leading to amino acidsubstitutions at “non-essential” amino acid residues can be made in thesequence of SEQ ID NO:1, 3, 4, 6, 7, 9, 12, 14, 15, 17, 19, 21, 27, 29,30, 32, 33, 35, 36, 38, 39, 41, 51, 53, 54, 56, 57, or 59. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence of MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5 (e.g., the sequences ofSEQ ID NO:2, 5, 8, 13, 16, 20, 28, 31, 34, 37, 40, 52, 55 and/or 58)without altering the biological activity, whereas an “essential” aminoacid residue is required for biological activity. For example, aminoacid residues that are conserved among the MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or the HST-5polypeptides of the present invention, e.g., those present in atransmembrane domain and/or a sugar transporter family domain, arepredicted to be particularly unamenable to alteration. Furthermore,additional amino acid residues that are conserved between the MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or the HST-5 polypeptides of the present invention and other membersof the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or the HST-5 family are not likely to be amenableto alteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding OAT, HST-1, or PLTR-1, proteins that contain changesin amino acid residues that are not essential for activity. Such OATproteins differ in amino acid sequence from SEQ ID NO:5, 8, 13, or 20,yet retain biological activity. In one embodiment, the isolated nucleicacid molecule comprises a nucleotide sequence encoding a protein,wherein the protein comprises an amino acid sequence at least about 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,99.9% or more identical to SEQ ID NO:5, 8, 13, or 20, e.g., to theentire length of SEQ ID NO:5, 8, 13, or 20.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding MTP-1, TP-2, TFM-2, TFM-3, 67118, 67067, 62092, HAAT,HST-4 and/or HST-5 polypeptides that contain changes in amino acidresidues that are not essential for activity. Such MTP-1, TP-2, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 polypeptides differin amino acid sequence from SEQ ID NO:2, 16, 28, 31, 34, 37, 40, 52, 55or 58, yet retain biological activity. In one embodiment, the isolatednucleic acid molecule comprises a nucleotide sequence encoding apolypeptide, wherein the polypeptide comprises an amino acid sequence atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:2, 16, 28,31, 34, 37, 40, 52, 55 or 58 (e.g., to the entire length of SEQ ID NO:2,16, 28, 31, 34, 37, 40, 52, 55 or 58).

An isolated nucleic acid molecule encoding an MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5polypeptide identical to the polypeptide of SEQ ID NO:2, 5, 8, 13, 16,20, 28, 31, 34, 37, 40, 52, 55 or 58, can be created by introducing oneor more nucleotide substitutions, additions or deletions into thenucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7, 9, 12, 14, 15, 17, 19,21, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 51, 53, 54, 56, 57, or 59,such that one or more amino acid substitutions, additions or deletionsare introduced into the encoded polypeptide. Mutations can be introducedinto SEQ ID NO:1, 3, 4, 6, 7, 9, 12, 14, 15, 17, 19, 21, 27, 29, 30, 32,33, 35, 36, 38, 39, 41, 51, 53, 54, 56, 57, or 59, by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in an MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5polypeptide is preferably replaced with another amino acid residue fromthe same side chain family. Alternatively, in another embodiment,mutations can be introduced randomly along all or part of an MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 coding sequence, such as by saturation mutagenesis, and theresultant mutants can be screened for MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 biologicalactivity to identify mutants that retain activity. Following mutagenesisof SEQ ID NO:1, 3, 4, 6, 7, 9, 12, 14, 15, 17, 19, 21, 27, 29, 30, 32,33, 35, 36, 38, 39, 41, 51, 53, 54, 56, 57, or 59, the encodedpolypeptide can be expressed recombinantly and the activity of thepolypeptide can be determined.

In a preferred embodiment, a mutant MTP-1 protein can be assayed for theability to metabolize or catabolize biochemical molecules necessary forenergy production or storage, permit intra- or intercellular signaling,metabolize or catabolize metabolically important biomolecules, and todetoxify potentially harmful compounds, or to facilitate thecompartmentalization of these molecules into a sequestered intracellularspace (e.g., the peroxisome).

In a preferred embodiment, a mutant OAT protein can be assayed for theability to (i) interact with an OAT substrate or target molecule; (ii)transport an OAT substrate across a membrane; (iii) interact with and/ormodulation of a second non-OAT protein; (iv) modulate cellular signalingand/or gene transcription (e.g., either directly or indirectly); (v)protect cells and/or tissues from organic anions; and/or (vi) modulatehormonal responses.

In a preferred embodiment, a mutant HST-1 polypeptide can be assayed forthe ability to (1) maintain sugar homeostasis in a cell, (2) influenceinsulin and/or glucagon secretion, (3) bind a monosaccharide, e.g.,D-glucose, D-fructose, and/or D-galactose, and (4) transportmonosaccharides across a cell membrane.

In a preferred embodiment, a mutant TP-2 polypeptide can be assayed forthe ability to 1) modulate the import and export of molecules, e.g.,hormones, ions, cytokines, neurotransmitters, monosaccharides, andmetabolites, from cells, 2) modulate intra- or inter-cellular signaling,3) modulate removal of potentially harmful compounds from the cell, orfacilitate the compartmentalization of these molecules into asequestered intra-cellular space (e.g., the peroxisome), and 4) modulatetransport of biological molecules across membranes, e.g., the plasmamembrane, or the membrane of the mitochondrion, the peroxisome, thelysosome, the endoplasmic reticulum, the nucleus, or the vacuole.

In a preferred embodiment, a mutant PLTR-1 protein can be assayed forthe ability to (i) interact with a PLTR-1 substrate or target molecule(e.g., a phospholipid, ATP, or a non-PLTR-1 protein); (ii) transport aPLTR-1 substrate or target molecule (e.g., an aminophospholipid such asphosphatidylserine or phosphatidylethanolamine) from one side of acellular membrane to the other; (iii) be phosphorylated ordephosphorylated; (iv) adopt an E1 conformation or an E2 conformation;(v) convert a PLTR-1 substrate or target molecule to a product (e.g.,hydrolysis of ATP); (vi) interact with a second non-PLTR-1 protein;(vii) modulate substrate or target molecule location (e.g., modulationof phospholipid location within a cell and/or location with respect to acellular membrane); (viii) maintain aminophospholipid gradients; (ix)modulate blood coagulation; (x) modulate intra- or intercellularsignaling and/or gene transcription (e.g., either directly orindirectly); and/or (xi) modulate cellular proliferation, growth,differentiation, apoptosis, absorption, or secretion.

In a preferred embodiment, a mutant TFM-2 and/or TFM-3 polypeptide canbe assayed for the ability to 1) modulate the import and export ofmolecules, e.g., hormones, ions, cytokines, neurotransmitters,monocarboxylates monosaccharides, and metabolites, from cells, 2)modulate intra- or inter-cellular signaling, 3) modulate removal ofpotentially harmful compounds from the cell, or facilitate thecompartmentalization of these molecules into a sequesteredintra-cellular space (e.g., the peroxisome), and 4) modulate transportof biological molecules across membranes, e.g., the plasma membrane, orthe membrane of the mitochondrion, the peroxisome, the lysosome, theendoplasmic reticulum, the nucleus, or the vacuole.

In a preferred embodiment, a mutant 67118 or 67067 polypeptide can beassayed for the ability to (i) interact with a 67118 or 67067 substrateor target molecule (e.g., a phospholipid, ATP, or a non-67118 or -67067protein); (ii) transport a 67118 or 67067 substrate or target molecule(e.g., an aminophospholipid such as phosphatidylserine orphosphatidylethanolamine) from one side of a cellular membrane to theother; (iii) be phosphorylated or dephosphorylated; (iv) adopt an Elconformation or an E2 conformation; (v) convert a 67118 or 67067substrate or target molecule to a product (e.g., hydrolysis of ATP);(vi) interact with a second non-67118 or -67067 protein; (vii) modulatesubstrate or target molecule location (e.g., modulation of phospholipidlocation within a cell and/or location with respect to a cellularmembrane); (viii) maintain aminophospholipid gradients; (ix) modulateintra- or intercellular signaling and/or gene transcription (e.g.,either directly or indirectly); and/or (x) modulate cellularproliferation, growth, differentiation, apoptosis, absorption, orsecretion.

In another preferred embodiment, a mutant 62092 protein can be assayedfor the ability to (i) interact with a 62092 substrate or targetmolecule (e.g., a nucleotide such as a purine mononucleotide or adinucleoside polyphosphate, or a non-62092 protein); (ii) convert a62092 substrate or target molecule to a product (e.g., cleave adinucleoside polyphosphate); (iii) interact with a second non-62092protein; (iv) sense of cellular stress signals; (v) regulate substrateor target molecule availability or activity; (vi) modulate intra- orintercellular signaling and/or gene transcription (e.g., either directlyor indirectly); and/or (vii) modulate cellular proliferation, growth,differentiation, and/or apoptosis.

In a preferred embodiment, a mutant HAAT protein can be assayed for theability to (i) interact with a HAAT substrate or target molecule (e.g.,an amino acid); (ii) transport a HAAT substrate or target molecule(e.g., an amino acid) from one side of a cellular membrane to the other;(iii) convert a HAAT substrate or target molecule to a product (e.g.,glucose production); (iv) interact with a second non-HAAT protein; (v)modulate substrate or target molecule location (e.g., modulation ofamino acid location within a cell and/or location with respect to acellular membrane); (vi) maintain amino acid gradients; (vii) modulatehormone metabolism and/or nerve transmission (e.g., either directly orindirectly); and/or (viii) modulate cellular proliferation, growth,differentiation, and production of metabolic energy.

In a preferred embodiment, a mutant HST-4 and/or HST-5 polypeptide canbe assayed for the ability to (1) bind a monosaccharide, e.g.,D-glucose, D-fructose, D-galactose, and/or mannose; (2) transportmonosaccharides across a cell membrane, (3) influence insulin and/orglucagon secretion; (4) maintain sugar homeostasis in a cell; and (5)mediate trans-epithelial movement in a cell.

In addition to the nucleic acid molecules encoding MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5 polypeptides described above, another aspect of the inventionpertains to isolated nucleic acid molecules which are antisense thereto.In an exemplary embodiment, the invention provides an isolated nucleicacid molecule which is antisense to an MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 nucleic acidmolecule (e.g., is antisense to the coding strand of an MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 nucleic acid molecule). An “antisense” nucleic acidcomprises a nucleotide sequence which is complementary to a “sense”nucleic acid encoding a polypeptide, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an mRNAsequence. Accordingly, an antisense nucleic acid can hydrogen bond to asense nucleic acid. The antisense nucleic acid can be complementary toan entire MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 coding strand, or to only a portionthereof. In one embodiment, an antisense nucleic acid molecule isantisense to a “coding region” of the coding strand of a nucleotidesequence encoding MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5. The term “coding region” refersto the region of the nucleotide sequence comprising codons which aretranslated into amino acid residues (e.g., the coding regions of humanMTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and HST-5 correspond to SEQ ID NO:3, 6, 9, 14, 17, 21, 29,32, 35, 38, 41, 53, 56, and 59, respectively). In another embodiment,the antisense nucleic acid molecule is antisense to a “noncoding region”of the coding strand of a nucleotide sequence encoding MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5. The term “noncoding region” refers to 5′ and 3′ sequenceswhich flank the coding region that are not translated into amino acids(i.e., also referred to as 5′ and 3′ untranslated regions).

Given the coding strand sequences encoding MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5disclosed herein (e.g., SEQ I) NO:3, 6, 9, 14, 17, 21, 29, 32, 35, 38,41, 53, 56, and 59, respectively), antisense nucleic acids of theinvention can be designed according to the rules of Watson and Crickbase pairing. The antisense nucleic acid molecule can be complementaryto the entire coding region of MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 mRNA, but morepreferably is an oligonucleotide which is antisense to only a portion ofthe coding or noncoding region of MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 mRNA. Forexample, the antisense oligonucleotide can be complementary to theregion surrounding the translation start site of MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5 mRNA (e.g., between the −10 and +10 regions of the start site of agene nucleotide sequence). An antisense oligonucleotide can be, forexample, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides inlength. An antisense nucleic acid of the invention can be constructedusing chemical synthesis and enzymatic ligation reactions usingprocedures known in the art. For example, an antisense nucleic acid(e.g., an antisense oligonucleotide) can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Examples of modifiednucleotides which can be used to generate the antisense nucleic acidinclude 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding an MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 polypeptide to thereby inhibit expression of thepolypeptide, e.g., by inhibiting transcription and/or translation. Thehybridization can be by conventional nucleotide complementarity to forma stable duplex, or, for example, in the case of an antisense nucleicacid molecule which binds to DNA duplexes, through specific interactionsin the major groove of the double helix. An example of a route ofadministration of antisense nucleic acid molecules of the inventioninclude direct injection at a tissue site. Alternatively, antisensenucleic acid molecules can be modified to target selected cells and thenadministered systemically. For example, for systemic administration,antisense molecules can be modified such that they specifically bind toreceptors or antigens expressed on a selected cell surface, e.g., bylinking the antisense nucleic acid molecules to peptides or antibodieswhich bind to cell surface receptors or antigens. The antisense nucleicacid molecules can also be delivered to cells using the vectorsdescribed herein. To achieve sufficient intracellular concentrations ofthe antisense molecules, vector constructs in which the antisensenucleic acid molecule is placed under the control of a strong pol II orpol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haseloff and Gerlach(1988) Nature 334:585-591)) can be used to catalytically cleave MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 mRNA transcripts to thereby inhibit translation of MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 mRNA. A ribozyme having specificity for an MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4-and/or HST-5-encoding nucleic acid can be designed based upon thenucleotide sequence of an MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5 cDNA disclosed herein(i.e., SEQ ID NO:1, 3, 4, 6, 7, 9, 12, 14, 15, 17, 19, 21, 27, 29, 30,32, 33, 35, 36, 38, 39, 41, 51, 53, 54, 56, 57, or 59). For example, aderivative of a Tetrahymena L-19 IVS RNA can be constructed in which thenucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in an MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4- and/or HST-5-encodingmRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al.U.S. Pat. No. 5,116,742. Alternatively, MTP-1, OAT, HST-1, TP-2,PLTR-.1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5mRNA can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules. See, e.g., Bartel,D. and Szostak, J. W. (1993) Science 261:1411-1418.

Alternatively, MTP-1 gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of the MTP-1(e.g., the MTP-1 promoter and/or enhancers; e.g., nucleotides 1-107 ofSEQ ID NO:1) to form triple helical structures that preventtranscription of the MTP-1 gene in target cells. See generally, Helene,C. (1991) Anticancer Drug Des. 6(6): 569-84; Helene, C. et al. (1992)Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays14(12):807-15.

Alternatively, OAT gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of the OAT(e.g., the OAT promoter and/or enhancers; e.g., nucleotides 1-371 of SEQID NO:4) to form triple helical structures that prevent transcription ofthe OAT gene in target cells. See generally, Helene, C. (1991)Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N.Y.Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioessays 14(12):807-15.

Alternatively, HST-1 gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of the HST-1(e.g., the HST-1 promoter and/or enhancers) to form triple helicalstructures that prevent transcription of the HST-1 gene in target cells.See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84;Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L.J. (1992) Bioassays 14(12):807-15.

Alternatively, TP-2 gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of the TP-2(e.g., the TP-2 promoter and/or enhancers) to form triple helicalstructures that prevent transcription of the TP-2 gene in target cells.See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84;Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L.J. (1992) Bioassays 14(12):807-15.

Alternatively, PLTR-1 gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of thePLTR-1 (e.g., the PLTR-1 promoter and/or enhancers; e.g., nucleotides1-170 of SEQ ID NO: 19) to form triple helical structures that preventtranscription of the PLTR-1 gene in target cells. See generally, Helene,C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992)Ann. N. Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioessays14(12):807-15.

Alternatively, TFM-2 and/or TFM-3 gene expression can be inhibited bytargeting nucleotide sequences complementary to the regulatory region ofthe TFM-2 and/or TFM-3 (e.g., the TFM-2 and/or TFM-3 promoter and/orenhancers) to form triple helical structures that prevent transcriptionof the TFM-2 and/or TFM-3 gene in target cells. See generally, Helene,C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992)Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays14(12):807-15.

Alternatively, 67118, 67067, and/or 62092 gene expression can beinhibited by targeting nucleotide sequences complementary to theregulatory region of the 67118, 67067, and/or 62092 (e.g., the 67118,67067, and/or 62092 promoter and/or enhancers) to form triple helicalstructures that prevent transcription of the 67118, 67067, and/or 62092gene in target cells. See generally, Helene, C. (1991) Anticancer DrugDes. 6(6):569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci.660:27-36; and Maher, L. J. (1992) Bioassays 14(12):807-15.

Alternatively, HAAT gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of the HAAT(e.g., the HAAT promoter and/or enhancers; e.g., nucleotides 1-68 of SEQID NO:51) to form triple helical structures that prevent transcriptionof the HAAT gene in target cells. See generally, Helene, C. (1991)Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N.Y.Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioessays 14(12):807-15.

Alternatively, HST-4 and/or HST-5 gene expression can be inhibited bytargeting nucleotide sequences complementary to the regulatory region ofthe HST-4 and/or HST-5 (e.g., the HST-4 and/or HST-5 promoter and/orenhancers) to form triple helical structures that prevent transcriptionof the HST-4 and/or HST-5 gene in target cells. See generally, Helene,C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992)Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays14(12):807-15.

In yet another embodiment, the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 nucleic acidmolecules of the present invention can be modified at the base moiety,sugar moiety or phosphate backbone to improve, e.g., the stability,hybridization, or solubility of the molecule. For example, thedeoxyribose phosphate backbone of the nucleic acid molecules can bemodified to generate peptide nucleic acids (see Hyrup B. et al. (1996)Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms“peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g.,DNA mimics, in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of PNAs has been shown to allow forspecific hybridization to DNA and RNA under conditions of low ionicstrength. The synthesis of PNA oligomers can be performed using standardsolid phase peptide synthesis protocols as described in Hyrup B. et al.(1996) supra; Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.

PNAs of MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 nucleic acid molecules can be used intherapeutic and diagnostic applications. For example, PNAs can be usedas antisense or antigene agents for sequence-specific modulation of geneexpression by, for example, inducing transcription or translation arrestor inhibiting replication. PNAs of MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 nucleic acidmolecules can also be used in the analysis of single base pair mutationsin a gene, (e.g., by PNA-directed PCR clamping); as ‘artificialrestriction enzymes’ when used in combination with other enzymes, (e.g.,S1 nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNAsequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefesupra).

In another embodiment, PNAs of MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 can be modified,(e.g., to enhance their stability or cellular uptake), by attachinglipophilic or other helper groups to PNA, by the formation of PNA-DNAchimeras, or by the use of liposomes or other techniques of drugdelivery known in the art. For example, PNA-DNA chimeras of MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 nucleic acid molecules can be generated which may combinethe advantageous properties of PNA and DNA. Such chimeras allow DNArecognition enzymes, (e.g., RNase H and DNA polymerases), to interactwith the DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup B. (1996) supra).The synthesis of PNA-DNA chimeras can be performed as described in HyrupB. (1996) supra and Finn P. J. et al. (1996) Nucleic Acids Res. 24 (17):3357-63. For example, a DNA chain can be synthesized on a solid supportusing standard phosphoramidite coupling chemistry and modifiednucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite, can be used as a between the PNA and the 5′ end of DNA(Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers arethen coupled in a stepwise manner to produce a chimeric molecule with a5′ PNA segment and a 3′ DNA segment (Finn P. J. et al. (1996) supra).Alternatively, chimeric molecules can be synthesized with a 5′ DNAsegment and a 3′ PNA segment (Peterser, K. H. et al. (1975) BioorganicMed. Chem. Lett. 5: 1119-11124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (See, e.g., Krolet al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, (e.g., a peptide,hybridization triggered cross-linking agent, transport agent, orhybridization-triggered cleavage agent).

Alternatively, the expression characteristics of an endogenous MTP-1,HST-1, TP-2, TFM-2, TFM-3, 67118, 67067, 62092, HST-4 and/or HST-5 genewithin a cell line or microorganism may be modified by inserting aheterologous DNA regulatory element into the genome of a stable cellline or cloned microorganism such that the inserted regulatory elementis operatively linked with the endogenous MTP-1, HST-1, TP-2, TFM-2,TFM-3, 67118, 67067, 62092, HST-4 and/or HST-5 gene. For example, anendogenous MTP-1, HST-1, TP-2, TFM-2, TFM-3, 67118, 67067, 62092, HST-4and/or HST-5 gene which is normally “transcriptionally silent”, i.e., anMTP-1, HST-1, TP-2, TFM-2, TFM-3, 67118, 67067, 62092, HST-4 and/orHST-5 gene which is normally not expressed, or is expressed only at verylow levels in a cell line or microorganism, may be activated byinserting a regulatory element which is capable of promoting theexpression of a normally expressed gene product in that cell line ormicroorganism. Alternatively, a transcriptionally silent, endogenousMTP-1, HST-1, TP-2, TFM-2, TFM-3, 67118, 67067, 62092, HST-4 and/orHST-5 gene may be activated by insertion of a promiscuous regulatoryelement that works across cell types.

A heterologous regulatory element may be inserted into a stable cellline or cloned microorganism, such that it is operatively linked with anendogenous MTP-1, HST-1, TP-2, TFM-2, TFM-3, 67118, 67067, 62092, HST-4and/or HST-5 gene, using techniques, such as targeted homologousrecombination, which are well known to those of skill in the art, anddescribed, e.g., in Chappel, U.S. Pat. No. 5,272,071; PCT publicationNo. WO 91/06667, published May 16, 1991.

II. Isolated Polypeptides and Antibodies

One aspect of the invention pertains to isolated or recombinant MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 proteins and polypeptides, and biologically active portionsthereof, as well as polypeptide fragments suitable for use as immunogensto raise anti-MTP-1, anti-OAT, anti-HST-1, anti-TP-2, anti-PLTR-1,anti-TFM-2, anti-TFM-3, anti-67118, anti-67067, anti-62092, anti-HAAT,anti-HST-4 and/or anti-HST-5 antibodies. In one embodiment, nativeMTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5 polypeptides can be isolated from cells ortissue sources by an appropriate purification scheme using standardprotein purification techniques. In another embodiment, MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 polypeptides are produced by recombinant DNA techniques.Alternative to recombinant expression, an MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5polypeptide or polypeptide can be synthesized chemically using standardpeptide synthesis techniques.

An “isolated” or “purified” polypeptide or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theMTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5 polypeptide is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesized.The language “substantially free of cellular material” includespreparations of MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 polypeptide in which thepolypeptide is separated from cellular components of the cells fromwhich it is isolated or recombinantly produced. In one embodiment, thelanguage “substantially free of cellular material” includes preparationsof MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5 polypeptide having less than about 30% (by dryweight) of non-MTP-1, non-OAT, non-HST-1, non-TP-2, non-PLTR-1,non-TFM-2, non-TFM-3, non-67118, non-67067, non-62092, non- HAAT,non-HST-4 and/or non-HST-5 polypeptide (also referred to herein as a“contaminating protein”), more preferably less than about 20% ofnon-MTP-1, non-OAT, non-HST-1, non-TP-2, non-PLTR-1, non-TFM-2,non-TFM-3, non-67118, non-67067, non-62092, non- HAAT, non-HST-4 and/ornon-HST-5 polypeptide, still more preferably less than about 10% ofnon-MTP-1, non-OAT, non-HST-1, non-TP-2, non-PLTR-1, non-TFM-2,non-TFM-3, non-67118, non-67067, non-62092, non- HAAT, non-HST-4 and/ornon-HST-5 polypeptide, and most preferably less than about 5% non-MTP-1,non-OAT, non-HST-1, non-TP-2, non-PLTR-1, non-TFM-2, non-TFM-3,non-67118, non-67067, non-62092, non- HAAT, non-HST-4 and/or non-HST-5polypeptide. When the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5 polypeptide orbiologically active portion thereof is recombinantly produced, it isalso preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, more preferably less than about10%, and most preferably less than about 5% of the volume of the proteinpreparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 polypeptidein which the polypeptide is separated from chemical precursors or otherchemicals which are involved in the synthesis of the polypeptide. In oneembodiment, the language “substantially free of chemical precursors orother chemicals” includes preparations of MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5polypeptide having less than about 30% (by dry weight) of chemicalprecursors or non-MTP-1, non-OAT, non-HST-1, non-TP-2, non-PLTR-1,non-TFM-2, non-TFM-3, non-67118, non-67067, non-62092, non-HAAT,non-HST-4 and/or non-HST-5 chemicals, more preferably less than about20% chemical precursors or non-MTP-1, non-OAT, non-HST-1, non-TP-2,non-PLTR-1, non-TFM-2, non-TFM-3, non-67118, non-67067, non-62092, non-HAAT, non-HST-4 and/or non-HST-5 chemicals, still more preferably lessthan about 10% chemical precursors or non-MTP-1, non-OAT, non-HST-1,non-TP-2, non-PLTR-1, non-TFM-2, non-TFM-3, non-67118, non-67067,non-62092, non- HAAT, non-HST-4 and/or non-HST-5 chemicals, and mostpreferably less than about 5% chemical precursors or non-MTP-1, non-OAT,non-HST-1, non-TP-2, non-PLTR-1, non-TFM-2, non-TFM-3, non-67118,non-67067, non-62092, non-HAAT, non-HST-4 and/or non-HST-5 chemicals.

As used herein, a “biologically active portion” of an MTP-1 proteinincludes a fragment of an MTP-1 protein which participates in aninteraction between an MTP-1 molecule and a non-MTP-1 molecule.Biologically active portions of an MTP-1 protein include peptidescomprising amino acid sequences sufficiently identical to or derivedfrom the amino acid sequence of the MTP-1 protein, e.g., the amino acidsequence shown in SEQ ID NO:2, which include less amino acids than thefull length MTP-1 protein, and exhibit at least one activity of an MTP-1protein. Typically, biologically active portions comprise a domain ormotif with at least one activity of the MTP-1 protein, e.g.,transporting a substrate molecule across a biological membrane. Abiologically active portion of an MTP-1 protein can be a polypeptidewhich is, for example, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300 ormore amino acids in length. Biologically active portions of an MTP-1protein can be used as targets for developing agents which modulate anMTP-1 mediated activity, e.g., lipid transport.

In one embodiment, a biologically active portion of an MTP-1 proteincomprises at least one transmembrane domain. It is to be understood thata preferred biologically active portion of an MTP-1 protein of thepresent invention may contain at least one transmembrane domain and oneor more of the following domains: a transmembrane domain, and/or an ABCtransporter domain. Moreover, other biologically active portions, inwhich other regions of the protein are deleted, can be prepared byrecombinant techniques and evaluated for one or more of the functionalactivities of a native MTP-1 protein.

In a preferred embodiment, the MTP-1 protein has an amino acid sequenceshown in SEQ ID NO:2. In other embodiments, the MTP-1 protein issubstantially identical to SEQ ID NO:2, and retains the functionalactivity of the protein of SEQ ID NO:2, yet differs in amino acidsequence due to natural allelic variation or mutagenesis, as describedin detail in subsection I above. Accordingly, in another embodiment, theMTP-1 protein is a protein which comprises an amino acid sequence atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or more identical to SEQ ID NO:2.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-identical sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence (e.g., when aligning a second sequence to the MTP-1 amino acidsequence of SEQ ID NO:2 having 400 amino acid residues, at least 50,preferably at least 100, more preferably at least 150, even morepreferably at least 200, and even more preferably at least 300 or moreamino acid residues are aligned). The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

As used herein, a “biologically active portion” of an OAT proteinincludes a fragment of an OAT protein which participates in aninteraction between an OAT molecule and a non-OAT molecule (e.g., an OATsubstrate or target molecule). Biologically active portions of an OATprotein include peptides comprising amino acid sequences sufficientlyhomologous to or derived from the OAT amino acid sequences, e.g., theamino acid sequences shown in SEQ ID NO:5 or 8, which include sufficientamino acid residues to exhibit at least one activity of an OAT protein.Typically, biologically active portions comprise a domain or motif withat least one activity of the OAT protein, e.g., OAT substratetransporting activity, OAT substrate or target molecule bindingactivity, intra- or inter-cellular signal modulating activity, geneexpression modulating activity, hormonal response modulating activity,and/or the ability to protect cells and/or tissues from organic anions.A biologically active portion of an OAT protein can be a polypeptidewhich is, for example, 10, 25, 50, 75, 100, 125, 150, 175, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700 or more amino acids inlength. Biologically active portions of an OAT protein can be used astargets for developing agents which modulate an OAT mediated activity,e.g., OAT substrate transport, OAT substrate or target molecule binding,intra- or inter-cellular signaling, cellular gene expression, hormonalresponses, and/or protection of cells and/or tissues from organicanions.

In one embodiment, a biologically active portion of an OAT proteincomprises at least one transmembrane domain. Moreover, otherbiologically active portions, in which other regions of the protein aredeleted, can be prepared by recombinant techniques and evaluated for oneor more of the functional activities of a native OAT protein.

Another aspect of the invention features fragments of the protein havingthe amino acid sequence of SEQ ID NO:5 or 8, for example, for use asimmunogens. In one embodiment, a fragment comprises at least 8 aminoacids (e.g., contiguous or consecutive amino acids) of the amino acidsequence of SEQ ID NO:5 or 8. In another embodiment, a fragmentcomprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more aminoacids (e.g., contiguous or consecutive amino acids) of the amino acidsequence of SEQ ID NO:5 or 8.

In a preferred embodiment, an OAT protein has an amino acid sequenceshown in SEQ ID NO:5 or 8. In other embodiments, the OAT protein issubstantially identical to SEQ ID NO:5 or 8, and retains the functionalactivity of the protein of SEQ ID NO:5 or 8, yet differs in amino acidsequence due to natural allelic variation or mutagenesis, as describedin detail in subsection I above. In another embodiment, the OAT proteinis a protein which comprises an amino acid sequence at least about 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,99.9% or more identical to SEQ ID NO:5 or 8.

In another embodiment, the invention features an OAT protein which isencoded by a nucleic acid molecule consisting of a nucleotide sequenceat least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,99.6%, 99.7%, 99.8%, 99.9% or more identical to a nucleotide sequence ofSEQ ID NO:4, 6, 7, or 9, or a complement thereof. This invention furtherfeatures an OAT protein which is encoded by a nucleic acid moleculeconsisting of a nucleotide sequence which hybridizes under stringenthybridization conditions to a complement of a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:4, 6, 7, or 9, or acomplement thereof.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence (e.g., when aligning a second sequence to the OAT amino acidsequence of SEQ ID NO:5 having 550 amino acid residues, at least 165,preferably at least 220, more preferably at least 275, even morepreferably at least 330, and even more preferably at least 385, 440 or495 amino acid residues are aligned; when aligning a second sequence tothe OAT amino acid sequence of SEQ ID NO:8 having 724 amino acidresidues, at least 217, preferably at least 290, more preferably atleast 362, even more preferably at least 434, and even more preferablyat least 507, 579 or 652 amino acid residues are aligned). The aminoacid residues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

As used herein, a “biologically active portion” of an HST-1 polypeptideincludes a fragment of an HST-1 polypeptide which participates in aninteraction between an HST-1 molecule and a non-HST-1 molecule.Biologically active portions of an HST-1 polypeptide include peptidescomprising amino acid sequences sufficiently identical to or derivedfrom the amino acid sequence of the HST-1 polypeptide, e.g., the aminoacid sequence shown in SEQ ID NO:13, which include less amino acids thanthe full length HST-1 polypeptides, and exhibit at least one activity ofan HST-1 polypeptide. Typically, biologically active portions comprise adomain or motif with at least one activity of the HST-1 polypeptide,e.g., modulating sugar transport mechanisms. A biologically activeportion of an HST-1 polypeptide can be a polypeptide which is, forexample, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 155, 175, 200, 225,250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550 or moreamino acids in length. Biologically active portions of an HST-1polypeptide can be used as targets for developing agents which modulatean HST-1 mediated activity, e.g., a sugar transport mechanism.

In one embodiment, a biologically active portion of an HST-1 polypeptidecomprises at least one transmembrane domain. It is to be understood thata preferred biologically active portion of an HST-1 polypeptide of thepresent invention comprises at least one or more of the followingdomains: a transmembrane domain and/or a sugar transporter familydomain. Moreover, other biologically active portions, in which otherregions of the polypeptide are deleted, can be prepared by recombinanttechniques and evaluated for one or more of the functional activities ofa native HST-1 polypeptide.

Another aspect of the invention features fragments of the polypeptidehaving the amino acid sequence of SEQ ID NO:13, for example, for use asimmunogens. In one embodiment, a fragment comprises at least 5 aminoacids (e.g., contiguous or consecutive amino acids) of the amino acidsequence of SEQ ID NO:13. In another embodiment, a fragment comprises atleast 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids (e.g.,contiguous or consecutive amino acids) of the amino acid sequence of SEQID NO:13.

In a preferred embodiment, an HST-1 polypeptide has an amino acidsequence shown in SEQ ID NO:13. In other embodiments, the HST-1polypeptide is substantially identical to SEQ ID NO:13, and retains thefunctional activity of the polypeptide of SEQ ID NO:13, yet differs inamino acid sequence due to natural allelic variation or mutagenesis, asdescribed in detail in subsection I above. In another embodiment, theHST-1 polypeptide is a polypeptide which comprises an amino acidsequence at least about 50%, 55%, 60%, 65%, 70%,75%, 80%,85%,90%,95%,96%,97%,98%,99%, 99.1%,99.2%, 99.3%,99.4%, 99.5%, 99.6%,99.7%, 99.8%, 99.9% or more identical to SEQ ID NO:13.

In another embodiment, the invention features an HST-1 polypeptide whichis encoded by a nucleic acid molecule consisting of a nucleotidesequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, 99.9% or more identical to a nucleotide sequence of SEQ ID NO:12or 14, or a complement thereof. This invention further features an HST-1polypeptide which is encoded by a nucleic acid molecule consisting of anucleotide sequence which hybridizes under stringent hybridizationconditions to a complement of a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:12 or 14, or a complement thereof.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-identical sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence (e.g., when aligning a second sequence to the HST-1 amino acidsequence of SEQ ID NO:13 having 419 amino acid residues, at least 126,preferably at least 168, more preferably at least 210, more preferablyat least 251, even more preferably at least 293, and even morepreferably at least 335 or 377 or more amino acid residues are aligned).The amino acid residues or nucleotides at corresponding amino acidpositions or nucleotide positions are then compared. When a position inthe first sequence is occupied by the same amino acid residue ornucleotide as the corresponding position in the second sequence, thenthe molecules are identical at that position (as used herein amino acidor nucleic acid “identity” is equivalent to amino acid or nucleic acid“homology”). The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.

As used herein, a “biologically active portion” of a TP-2 polypeptideincludes a fragment of a TP-2 polypeptide which participates in aninteraction between a TP-2 molecule and a non-TP-2 molecule.Biologically active portions of a TP-2 polypeptide include peptidescomprising amino acid sequences sufficiently identical to or derivedfrom the amino acid sequence of the TP-2 polypeptide, e.g., the aminoacid sequence shown in SEQ ID NO:16, which include less amino acids thanthe full length TP-2 polypeptides, and exhibit at least one activity ofa TP-2 polypeptide. Typically, biologically active portions comprise adomain or motif with at least one activity of the TP-2 polypeptide,e.g., modulating transport mechanisms. A biologically active portion ofa TP-2 polypeptide can be a polypeptide which is, for example, 25, 30,35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325,350, 375, 400, 425, 450, 475 or more amino acids in length. Biologicallyactive portions of a TP-2 polypeptide can be used as targets fordeveloping agents which modulate a TP-2 mediated activity, e.g.,modulating transport of biological molecules across membranes.

In one embodiment, a biologically active portion of a TP-2 polypeptidecomprises at least one transmembrane domain. It is to be understood thata preferred biologically active portion of a TP-2 polypeptide of thepresent invention comprises at least one or more of the followingdomains: a transmembrane domain, and/or a sugar transporter domain,and/or a LacY proton/sugar symporter domain. Moreover, otherbiologically active portions, in which other regions of the polypeptideare deleted, can be prepared by recombinant techniques and evaluated forone or more of the functional activities of a native TP-2 polypeptide.

Another aspect of the invention features fragments of the polypeptidehaving the amino acid sequence of SEQ ID NO:16, for example, for use asimmunogens. In one embodiment, a fragment comprises at least 5 aminoacids (e.g., contiguous or consecutive amino acids) of the amino acidsequence of SEQ ID NO:16. In another embodiment, a fragment comprises atleast 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids (e.g.,contiguous or consecutive amino acids) of the amino acid sequence of SEQID NO:16.

In a preferred embodiment, a TP-2 polypeptide has an amino acid sequenceshown in SEQ ID NO:16. In other embodiments, the TP-2 polypeptide issubstantially identical to SEQ ID NO:16, and retains the functionalactivity of the polypeptide of SEQ ID NO:16, yet differs in amino acidsequence due to natural allelic variation or mutagenesis, as describedin detail in subsection I above. In another embodiment, the TP-2polypeptide is a polypeptide which comprises an amino acid sequence atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or more identical to SEQ ID NO:16.

In another embodiment, the invention features a TP-2 polypeptide whichis encoded by a nucleic acid molecule consisting of a nucleotidesequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or more indentical to a nucleotide sequence ofSEQ ID NO:15 or 17, or a complement thereof. This invention furtherfeatures a TP-2 polypeptide which is encoded by a nucleic acid moleculeconsisting of a nucleotide sequence which hybridizes under stringenthybridization conditions to a complement of a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:15 or 17, or acomplement thereof.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-identical sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence (e.g., when aligning a second sequence to the TP-2 amino acidsequence of SEQ ID NO:16 having 474 amino acid residues, at least 142,preferably at least 189, more preferably at least 237, more preferablyat least 284, even more preferably at least 331, and even morepreferably at least 379 or 426 or more amino acid residues are aligned).The amino acid residues or nucleotides at corresponding amino acidpositions or nucleotide positions are then compared. When a position inthe first sequence is occupied by the same amino acid residue ornucleotide as the corresponding position in the second sequence, thenthe molecules are identical at that position (as used herein amino acidor nucleic acid “identity” is equivalent to amino acid or nucleic acid“homology”). The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.

As used herein, a “biologically active portion” of a PLTR-1 proteinincludes a fragment of a PLTR-1 protein which participates in aninteraction between a PLTR-1 molecule and a non-PLTR-1 molecule (e.g., aPLTR-1 substrate such as a phospholipid or ATP). Biologically activeportions of a PLTR-1 protein include peptides comprising amino acidsequences sufficiently homologous to or derived from the PLTR-1 aminoacid sequences, e.g., the amino acid sequences shown in SEQ ID NO:20,which include sufficient amino acid residues to exhibit at least oneactivity of a PLTR-1 protein. Typically, biologically active portionscomprise a domain or motif with at least one activity of the PLTR-1protein, e.g., the ability to interact with a PLTR-1 substrate or targetmolecule (e.g., a phospholipid; ATP; a non-PLTR-1 protein; or anotherPLTR-1 protein or subunit); the ability to transport a PLTR-1 substrateor target molecule (e.g., a phospholipid) from one side of a cellularmembrane to the other; the ability to be phosphorylated ordephosphorylated; the ability to adopt an E1 conformation or an E2conformation; the ability to convert a PLTR-1 substrate or targetmolecule to a product (e.g., the ability to hydrolyze ATP); the abilityto interact with a second non-PLTR-1 protein; the ability to modulateintra- or inter-cellular signaling and/or gene transcription (e.g.,either directly or indirectly); the ability to modulate cellular growth,proliferation, differentiation, absorption, and/or secretion. Abiologically active portion of a PLTR-1 protein can be a polypeptidewhich is, for example, 10, 15, 20, 25, 30, 25, 40, 45, 50, 75, 100, 125,150, 175, 200, 250, 300, 328, 350, 375, 400, 450, 465, 500, 520, 550,600, 650, 700, 703, 750, 800, 850, 900, 932, 950, 1000, 1050, 1100, 1150or more amino acids in length. Biologically active portions of a PLTR-1protein can be used as targets for developing agents which modulate aPLTR-1 mediated activity, e.g., any of the aforementioned PLTR-1activities.

In one embodiment, a biologically active portion of a PLTR-1 proteincomprises at least one at least one or more of the following domains,sites, or motifs: a transmembrane domain, an N-terminal largeextramembrane domain, a C-terminal large extramembrane domain, an E1-E2ATPases phosphorylation site, a P-type ATPase sequence 1 motif, a P-typeATPase sequence 2 motif, a P-type ATPase sequence 3 motif, and/or one ormore phospholipid transporter specific amino acid resides. Moreover,other biologically active portions, in which other regions of theprotein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of a nativePLTR-1 protein.

Another aspect of the invention features fragments of the protein havingthe amino acid sequence of SEQ ID NO:20, for example, for use asimmunogens. In one embodiment, a fragment comprises at least 5 aminoacids (e.g., contiguous or consecutive amino acids) of the amino acidsequence of SEQ ID NO:20. In another embodiment, a fragment comprises atleast 8, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids (e.g.,contiguous or consecutive amino acids) of the amino acid sequence of SEQID NO:20.

In a preferred embodiment, a PLTR-1 protein has an amino acid sequenceshown in SEQ ID NO:20. In other embodiments, the PLTR-1 protein issubstantially identical to SEQ ID NO:20, and retains the functionalactivity of the protein of SEQ ID NO:20, yet differs in amino acidsequence due to natural allelic variation or mutagenesis, as describedin detail in subsection I above. In another embodiment, the PLTR-1protein is a protein which comprises an amino acid sequence at leastabout 75%, 79%, 80%, 81%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or more identical to SEQ ID NO:20.

In another embodiment, the invention features a PLTR-1 protein which isencoded by a nucleic acid molecule consisting of a nucleotide sequenceat least about 75%, 79%, 80%, 81%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, 99.9% or more identical to a nucleotide sequence of SEQ ID NO:19or 21, or a complement thereof. This invention further features a PLTR-1protein which is encoded by a nucleic acid molecule consisting of anucleotide sequence which hybridizes under stringent hybridizationconditions to a complement of a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:19 or 21, or a complement thereof.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence (e.g., when aligning a second sequence to the PLTR-1 amino acidsequence of SEQ ID NO:20 having 1190 amino acid residues, at least 357,preferably at least 476, more preferably at least 595, even morepreferably at least 714, and even more preferably at least 833, 952 or1071 amino acid residues are aligned). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

As used herein, a “biologically active portion” of a TFM-2 and/or TFM-3polypeptide includes a fragment of a TFM-2 and/or TFM-3 polypeptidewhich participates in an interaction between a TFM-2 and/or TFM-3molecule and a non-TFM-2 and/or TFM-3 molecule. Biologically activeportions of a TFM-2 and/or TFM-3 polypeptide include peptides comprisingamino acid sequences sufficiently identical to or derived from the aminoacid sequence of the TFM-2 and/or TFM-3 polypeptide, e.g., the aminoacid sequence shown in SEQ ID NO:28 or 31, which include less aminoacids than the full length TFM-2 and/or TFM-3 polypeptides, and exhibitat least one activity of a TFM-2 and/or TFM-3 polypeptide. Typically,biologically active portions comprise a domain or motif with at leastone activity of the TFM-2 and/or TFM-3 polypeptide, e.g., modulatingtransport mechanisms. A biologically active portion of a TFM-2 and/orTFM-3 polypeptide can be a polypeptide which is, for example, 25, 30,35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325,350, 375 or more amino acids in length. Biologically active portions ofa TFM-2 and/or TFM-3 polypeptide can be used as targets for developingagents which modulate a TFM-2 and/or TFM-3 mediated activity, e.g.,modulating transport of biological molecules across membranes.

In one embodiment, a biologically active portion of a TFM-2 and/or TFM-3polypeptide comprises at least one transmembrane domain. It is to beunderstood that a preferred biologically active portion of a TFM-2and/or TFM-3 polypeptide of the present invention comprises at least oneor more of the following domains: a transmembrane domain, and/or amonocarboxylate domain, and/or a sugar transporter domain. Moreover,other biologically active portions, in which other regions of thepolypeptide are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of a native TFM-2and/or TFM-3 polypeptide.

Another aspect of the invention features fragments of the polypeptidehaving the amino acid sequence of SEQ ID NO:28 or 31, for example, foruse as immunogens. In one embodiment, a fragment comprises at least 5amino acids (e.g., contiguous or consecutive amino acids) of the aminoacid sequence of SEQ ID NO:28 or 31. In another embodiment, a fragmentcomprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more aminoacids (e.g., contiguous or consecutive amino acids) of the amino acidsequence of SEQ ID NO:28 or 31.

In a preferred embodiment, a TFM-2 and/or TFM-3 polypeptide has an aminoacid sequence shown in SEQ ID NO:28 or 31. In other embodiments, theTFM-2 and/or TFM-3 polypeptide is substantially identical to SEQ IDNO:28 or 31, and retains the functional activity of the polypeptide ofSEQ ID NO:28 or 31, yet differs in amino acid sequence due to naturalallelic variation or mutagenesis, as described in detail in subsection Iabove. In another embodiment, the TFM-2 and/or TFM-3 polypeptide is apolypeptide which comprises an amino acid sequence at least about 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or moreidentical to SEQ ID NO:28 or 31.

In another embodiment, the invention features a TFM-2 and/or TFM-3polypeptide which is encoded by a nucleic acid molecule consisting of anucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to a nucleotidesequence of SEQ ID NO:27, 29, 30, or 32, or a complement thereof. Thisinvention further features a TFM-2 and/or TFM-3 polypeptide which isencoded by a nucleic acid molecule consisting of a nucleotide sequencewhich hybridizes under stringent hybridization conditions to acomplement of a nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO:27, 29, 30, or 32, or a complement thereof.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-identical sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence (e.g., when aligning a second sequence to the TFM-2 amino acidsequence of SEQ ID NO:28 having 392 amino acid residues, at least 117,preferably at least 156, more preferably at least 196, more preferablyat least 235, even more preferably at least 274, and even morepreferably at least 313 or 352 or more amino acid residues are aligned;when aligning a second sequence to the TFM-3 amino acid sequence of SEQiID NO:31 having 405 amino acid residues, at least 121, preferably atleast 162, more preferably at least 202, more preferably at least 243,even more preferably at least 283, and even more preferably at least 324or 364 or more amino acid residues are aligned). The amino acid residuesor nucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

As used herein, a “biologically active portion” of a 67118, 67067,and/or 62092 polypeptide includes a fragment of a 67118, 67067, and/or62092 polypeptide which participates in an interaction between a 67118,67067, and/or 62092 molecule and a non-67118, 67067, and/or 62092molecule (e.g., a 67118 or 67067 substrate such as a phospholipid orATP, or a 62092 substrate such as a nucleotide or a non-62092 protein).Biologically active portions of a 67118, 67067, and/or 62092 polypeptideinclude peptides comprising amino acid sequences sufficiently identicalto or derived from the amino acid sequence of the 67118, 67067, and/or62092 polypeptide, e.g., the amino acid sequence shown in SEQ ID NO:34,37, or 40, which include less amino acids than the full length 67118,67067, and/or 62092 polypeptides, and exhibit at least one activity of a67118, 67067, and/or 62092 polypeptide.

Typically, biologically active portions of a 67118 or 67067 polypeptidecomprise a domain or motif with at least one activity of the 67118 or67067 polypeptide, e.g., the ability to interact with a 67118 or 67067substrate or target molecule (e.g., a phospholipid; ATP; a non-67118 or67067 protein; or another 67118 or 67067 protein or subunit); theability to transport a 67118 or 67067 substrate or target molecule(e.g., a phospholipid) from one side of a cellular membrane to theother; the ability to be phosphorylated or dephosphorylated; the abilityto adopt an E1 conformation or an E2 conformation; the ability toconvert a 67118 or 67067 substrate or target molecule to a product(e.g., the ability to hydrolyze ATP); the ability to interact with asecond non-67118 or 67067 protein; the ability to modulate intra- orinter-cellular signaling and/or gene transcription (e.g., eitherdirectly or indirectly); the ability to modulate cellular growth,proliferation, differentiation, absorption, and/or secretion. Abiologically active portion of a 67118 or 67067 polypeptide can be apolypeptide which is, for example, 10, 25, 50, 75, 100, 125, 150, 175,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450,1500, 1550 or more amino acids in length. Biologically active portionsof a 67118 or 67067 polypeptide can be used as targets for developingagents which modulate a 67118 or 67067 mediated activity, e.g.,modulating transport of biological molecules across membranes.

Moreover, biologically active portions of a 62092 protein typicallycomprise a domain or motif with at least one activity of the 62092protein, e.g., 62092 activity, nucleotide-binding activity, ability tomodulate intra- or inter-cellular signaling and/or gene expression,and/or ability to modulate cell growth, proliferation, differentiation,and/or apoptosis mechanisms. A biologically active portion of a 62092protein can be a polypeptide which is, for example, 10, 25, 50, 75, 100,125, 150 or more amino acids in length. Biologically active portions ofa 62092 protein can be used as targets for developing agents whichmodulate a 62092 mediated activity, e.g., 62092 activity,nucleotide-binding activity, ability to modulate intra- orinter-cellular signaling and/or gene expression, and/or ability tomodulate cell growth, proliferation, differentiation, and/or apoptosismechanisms.

In one embodiment, a biologically active portion of a 67118, or 67067polypeptide comprises at least one at least one or more of the followingdomains, sites, or motifs: a transmembrane domain, an N-terminal largeextramembrane domain, a C-terminal large extramembrane domain, an E1-E2ATPases phosphorylation site, a P-type ATPase sequence 1 motif, a P-typeATPase sequence 2 motif, a P-type ATPase sequence 3 motif, and/or one ormore phospholipid transporter specific amino acid resides. Moreover,other biologically active portions, in which other regions of thepolypeptide are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of a native67118, or 67067 polypeptide.

In another embodiment, a biologically active portion of a 62092 proteincomprises at least a 62092 family domain and/or a 62092 family signaturemotif. Moreover, other biologically active portions, in which otherregions of the protein are deleted, can be prepared by recombinanttechniques and evaluated for one or more of the functional activities ofa native 62092 protein.

Another aspect of the invention features fragments of the polypeptidehaving the amino acid sequence of SEQ ID NO:34, 37, or 40, for example,for use as immunogens. In one embodiment, a fragment comprises at least5 amino acids (e.g., contiguous or consecutive amino acids) of the aminoacid sequence of SEQ ID NO:34, 37, or 40. In another embodiment, afragment comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or moreamino acids (e.g., contiguous or consecutive amino acids) of the aminoacid sequence of SEQ ID NO:34, 37, or 40.

In a preferred embodiment, a 67118, 67067, and/or 62092 polypeptide hasan amino acid sequence shown in SEQ ID NO:34, 37, or 40. In otherembodiments, the 67118, 67067, and/or 62092 polypeptide is substantiallyidentical to SEQ ID NO:34, 37, or 40, and retains the functionalactivity of the polypeptide of SEQ ID NO:34, 37, or 40, yet differs inamino acid sequence due to natural allelic variation or mutagenesis, asdescribed in detail in subsection I above. In another embodiment, the67118, 67067, and/or 62092 polypeptide is a polypeptide which comprisesan amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:34, 37,or 40.

In another embodiment, the invention features a 67118, 67067, and/or62092 polypeptide which is encoded by a nucleic acid molecule consistingof a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical to anucleotide sequence of SEQ ID NO:33, 35, 36, 38, 39, or 41, or acomplement thereof. This invention further features a 67118, 67067,and/or 62092 polypeptide which is encoded by a nucleic acid moleculeconsisting of a nucleotide sequence which hybridizes under stringenthybridization conditions to a complement of a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:33, 35, 36, 38, 39, or41, or a complement thereof.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-identical sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence (e.g., when aligning a second sequence to the 67118 amino acidsequence of SEQ ID NO:34 having 1134 amino acid residues, at least 340,preferably at least 453, more preferably at least 567, more preferablyat least 640, even more preferably at least 793, and even morepreferably at least 907 or 1020 or more amino acid residues are aligned;when aligning a second sequence to the 67067 amino acid sequence of SEQID NO:37 having 1588 amino acid residues, at least 476, preferably atleast 635, more preferably at least 794, more preferably at least 952,even more preferably at least 1111, and even more preferably at least1270 or 1429 or more amino acid residues are aligned; when aligning asecond sequence to the 62092 amino acid sequence of SEQ ID NO:40 having163 amino acid residues, at least 48, preferably at least 65, morepreferably at least 81, more preferably at least 97, even morepreferably at least 114, and even more preferably at least 130 or 146 ormore amino acid residues are aligned). In another preferred embodiment,the sequences being aligned for comparison purposes are globally alignedand percent identity is determined over the entire length of thesequences aligned. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

As used herein, a “biologically active portion” of a HAAT proteinincludes a fragment of a HAAT protein which participates in aninteraction between a HAAT molecule and a non-HAAT molecule (e.g., aHAAT substrate such as an amino acid). Biologically active portions of aHAAT protein include peptides comprising amino acid sequencessufficiently homologous to or derived from the HAAT amino acidsequences, e.g., the amino acid sequences shown in SEQ ID NO:52, whichinclude sufficient amino acid residues to exhibit at least one activityof a HAAT protein. Typically, biologically active portions comprise adomain or motif with at least one activity of the HAAT protein, e.g.,(i) interaction with a HAAT substrate or target molecule (e.g., an aminoacid); (ii) transport of a HAAT substrate or target molecule (e.g., anamino acid) from one side of a cellular membrane to the other; (iii)conversion of a HAAT substrate or target molecule to a product (e.g.,glucose production); (iv) interaction with a second non-HAAT protein;(v) modulation of substrate or target molecule location (e.g.,modulation of amino acid location within a cell and/or location withrespect to a cellular membrane); (vi) maintenance of amino acidgradients; (vii) modulation of hormone metabolism and/or nervetransmission (e.g., either directly or indirectly); (viii) modulation ofcellular proliferation, growth, differentiation, and production ofmetabolic energy; and/or (ix) modulation of amino acid homeostasis. Abiologically active portion of a HAAT protein can be a polypeptide whichis, for example, 10, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350,400, 450, 475, or 485 or more amino acids in length. Biologically activeportions of a HAAT protein can be used as targets for developing agentswhich modulate a HAAT mediated activity, e.g., any of the aforementionedHAAT activities.

In one embodiment, a biologically active portion of a HAAT proteincomprises at least one at least one or more of the following domains,sites, or motifs: a transmembrane domain, a transmembrane amino acidtransporter domain, and/or one or more amino acid transporter specificamino acid residues. Moreover, other biologically active portions, inwhich other regions of the protein are deleted, can be prepared byrecombinant techniques and evaluated for one or more of the functionalactivities of a native HAAT protein.

Another aspect of the invention features fragments of the protein havingthe amino acid sequence of SEQ ID NO:52, for example, for use asimmunogens. In one embodiment, a fragment comprises at least 5 aminoacids (e.g., contiguous or consecutive amino acids) of the amino acidsequence of SEQ ID NO:52. In another embodiment, a fragment comprises atleast 8, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids (e.g.,contiguous or consecutive amino acids) of the amino acid sequence of SEQID NO:52.

In a preferred embodiment, a HAAT protein has an amino acid sequenceshown in SEQ ID NO:52. In other embodiments, the HAAT protein issubstantially identical to SEQ ID NO:52, and retains the functionalactivity of the protein of SEQ ID NO:52, yet differs in amino acidsequence due to natural allelic variation or mutagenesis, as describedin detail in subsection I above. In another embodiment, the HAAT proteinis a protein which comprises an amino acid sequence at least about 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ IDNO:52.

In another embodiment, the invention features a HAAT protein which isencoded by a nucleic acid molecule consisting of a nucleotide sequenceat least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or moreidentical to a nucleotide sequence of SEQ ID NO:51 or 53, or acomplement thereof. This invention further features a HAAT protein whichis encoded by a nucleic acid molecule consisting of a nucleotidesequence which hybridizes under stringent hybridization conditions to acomplement of a nucleic acid molecule comprising the nucleotide sequenceof SEQ I) NO:51 or 53, or a complement thereof.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence (e.g., when aligning a second sequence to the HAAT amino acidsequence of SEQ ID NO:52 having 485 amino acid residues, at least 157,preferably at least 276, more preferably at least 395, and even morepreferably at least 414 amino acid residues are aligned). The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein, amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

As used herein, a “biologically active portion” of an HST-4 and/or anHST-5 polypeptide includes a fragment of an HST-4 and/or an HST-5polypeptide which participates in an interaction between an HST-4 and/oran HST-5 molecule and a non-HST-4 and/or a non-HST-5 molecule.Biologically active portions of an HST-4 and/or an HST-5 polypeptideinclude peptides comprising amino acid sequences sufficiently identicalto or derived from the amino acid sequence of the HST-4 and/or the HST-5polypeptide, e.g., the amino acid sequence shown in SEQ ID NO:55 or 58,which include less amino acids than the full length HST-4 and/or HST-5polypeptides, and exhibit at least one activity of an HST-4 and/or anHST-5 polypeptide. Typically, biologically active portions comprise adomain or motif with at least one activity of the HST-4 and/or the HST-5polypeptide, e.g., modulating sugar transport mechanisms. A biologicallyactive portion of an HST-4 polypeptide can be a polypeptide which is,for example, 25, 30, 35, 40,-45, 50, 75, 100, 125, 150, 175, 200, 225,250, 275, 300, 325, 350, 375, 400, 425 or more amino acids in length. Abiologically active portion of an HST-5 polypeptide can be a polypeptidewhich is, for example, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 400, 425 or more amino acids inlength. Biologically active portions of an HST-4 and/or an HST-5polypeptide can be used as targets for developing agents which modulatean HST-4 and/or HST-5 mediated activity, e.g., a sugar transportmechanism.

In one embodiment, a biologically active portion of an HST-4 and/or anHST-5 polypeptide comprises at least one transmembrane domain. It is tobe understood that a preferred biologically active portion of an HST-4and/or an HST-5 polypeptide of the present invention comprises at leastone or more of the following domains: a transmembrane domain and/or asugar transporter family domain. Moreover, other biologically activeportions, in which other regions of the polypeptide are deleted, can beprepared by recombinant techniques and evaluated for one or more of thefunctional activities of a native HST-4 and/or HST-5 polypeptide.

Another aspect of the invention features fragments of the polypeptidehaving the amino acid sequence of SEQ ID NO:55 or 58, for example, foruse as immunogens. In one embodiment, a fragment comprises at least 5amino acids (e.g., contiguous or consecutive amino acids) of the aminoacid sequences of SEQ ID NO:55 or 58. In another embodiment, a fragmentcomprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more aminoacids (e.g., contiguous or consecutive amino acids) of the amino acidsequence of SEQ ID NO:55 or 58.

In a preferred embodiment, an HST-4 and/or an HST-5 polypeptide has anamino acid sequence shown in SEQ ID NO:55 or 58. In other embodiments,the HST-4 and/or the HST-5 polypeptide is substantially identical to SEQID NO:55 or 58, and retains the functional activity of the polypeptideof SEQ ID NO:55 or 58, yet differs in amino acid sequence due to naturalallelic variation or mutagenesis, as described in detail in subsection Iabove. In another embodiment, the HST-4 and/or the HST-5 polypeptide isa polypeptide which comprises an amino acid sequence at least about 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to SEQ ID NO:55 or 58.

In another embodiment, the invention features an HST-4 and/or an HST-5polypeptide which is encoded by a nucleic acid molecule consisting of anucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto a nucleotide sequence of SEQ ID NO:54, 56, 57, or 59, or a complementthereof. This invention further features an HST-4 and/or an HST-5polypeptide which is encoded by a nucleic acid molecule consisting of anucleotide sequence which hybridizes under stringent hybridizationconditions to a complement of a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:54, 56, 57, or 59, or a complementthereof.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-identical sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence (e.g., when aligning a second sequence to the HST-4 amino acidsequence of SEQ ID NO:55 having 438 amino acid residues, at least 131,preferably at least 175, more preferably at least 219, more preferablyat least 262, even more preferably at least 306, and even morepreferably at least 350 or 394 or more amino acid residues are aligned;when aligning a second sequence to the HST-5 amino acid sequence of SEQID NO:58 having 436 amino acid residues, at least 130, preferably atleast 174, more preferably at least 218, more preferably at least 261,even more preferably at least 305, and even more preferably at least 348or 392 or more amino acid residues are aligned). The amino acid residuesor nucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6. In yet another preferred embodiment, the percentidentity between two nucleotide sequences is determined using the GAPprogram in the GCG software package (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. A preferred, non-limitingexample of parameters to be used in conjunction with the GAP programinclude a Blosum 62 scoring matrix with a gap penalty of 12, a gapextend penalty of 4, and a frameshift gap penalty of 5.

In another embodiment, the percent identity between two amino acid ornucleotide sequences is determined using the algorithm of E. Meyers andW. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has beenincorporated into the ALIGN program (version 2.0 or version 2.0U), usinga PAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4.

The nucleic acid and polypeptide sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to HST-4 and/or HST-5 nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=100, wordlength=3, and a Blosum62 matrix to obtain aminoacid sequences homologous to HST-4 and/or HST-5 polypeptide molecules ofthe invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al., (1997)Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See the internet website for theNational Center for Biotechnology Information.

The invention also provides MTP-1 chimeric or fusion proteins. As usedherein, an MTP-1 “chimeric protein” or “fusion protein” comprises anMTP-1 polypeptide operatively linked to a non-MTP-1 polypeptide. An“MTP-1 polypeptide” refers to a polypeptide having an amino acidsequence corresponding to an MTP-1 molecule, whereas a “non-MTP-1polypeptide” refers to a polypeptide having an amino acid sequencecorresponding to a protein which is not substantially homologous to theMTP-1 protein, e.g., a protein which is different from the MTP-1 proteinand which is derived from the same or a different organism. Within anMTP-1 fusion protein the MTP-1 polypeptide can correspond to all or aportion of an MTP-1 protein. In a preferred embodiment, an MTP-1 fusionprotein comprises at least one biologically active portion of an MTP-1protein. In another preferred embodiment, an MTP-1 fusion proteincomprises at least two biologically active portions of an MTP-1 protein.Within the fusion protein, the term “operatively linked” is intended toindicate that the MTP-1 polypeptide and the non-MTP-1 polypeptide arefused in-frame to each other. The non-MTP-1 polypeptide can be fused tothe N-terminus or C-terminus of the MTP-1 polypeptide.

For example, in one embodiment, the fusion protein is a GST-MTP-1 fusionprotein in which the MTP-1 sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant MTP-1.

In another embodiment, the fusion protein is an MTP-1 protein containinga heterologous signal sequence at its N-terminus. In certain host cells(e.g., mammalian host cells), expression and/or secretion of MTP-1 canbe increased through use of a heterologous signal sequence.

The invention also provides OAT chimeric or fusion proteins. As usedherein, an OAT “chimeric protein” or “fusion protein” comprises an OATpolypeptide operatively linked to a non-OAT polypeptide. AN “OATpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to an OAT protein, whereas a “non-OAT polypeptide” refersto a polypeptide having an amino acid sequence corresponding to aprotein which is not substantially homologous to the OAT protein, e.g.,a protein which is different from the OAT protein and which is derivedfrom the same or a different organism. Within an OAT fusion protein theOAT polypeptide can correspond to all or a portion of an OAT protein. Ina preferred embodiment, an OAT fusion protein comprises at least onebiologically active portion of an OAT protein. In another preferredembodiment, an OAT fusion protein comprises at least two biologicallyactive portions of an OAT protein. Within the fusion protein, the term“operatively linked” is intended to indicate that the OAT polypeptideand the non-OAT polypeptide are fused in-frame to each other. Thenon-OAT polypeptide can be fused to the N-terminus or C-terminus of theOAT polypeptide.

For example, in one embodiment, the fusion protein is a GST-OAT fusionprotein in which the OAT sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant OAT. In another embodiment, the fusion protein is an OATprotein containing a heterologous signal sequence at its N-terminus. Incertain host cells (e.g., mammalian host cells), expression and/orsecretion of OAT can be increased through use of a heterologous signalsequence.

The invention also provides HST-1 chimeric or fusion proteins. As usedherein, an HST-1 “chimeric protein” or “fusion protein” comprises anHST-1 polypeptide operatively linked to a non-HST-1 polypeptide. An“HST-1 polypeptide” refers to a polypeptide having an amino acidsequence corresponding to HST-1, whereas a “non-HST-1 polypeptide”refers to a polypeptide having an amino acid sequence corresponding to apolypeptide which is not substantially homologous to the HST-1polypeptide, e.g., a polypeptide which is different from the HST-1polypeptide and which is derived from the same or a different organism.Within an HST-1 fusion protein the HST-1 polypeptide can correspond toall or a portion of an HST-1 polypeptide. In a preferred embodiment, anHST-1 fusion protein comprises at least one biologically active portionof an HST-1 polypeptide. In another preferred embodiment, an HST-1fusion protein comprises at least two biologically active portions of anHST-1 polypeptide. Within the fusion protein, the term “operativelylinked” is intended to indicate that the HST-1 polypeptide and thenon-HST-1 polypeptide are fused in-frame to each other. The non-HST-1polypeptide can be fused to the N-terminus or C-terminus of the HST-1polypeptide.

For example, in one embodiment, the fusion protein is a GST-HST-1 fusionprotein in which the HST-1 sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant HST-1.

In another embodiment, the fusion protein is an HST-1 polypeptidecontaining a heterologous signal sequence at its N-terminus. In certainhost cells (e.g., mammalian host cells), expression and/or secretion ofHST-1 can be increased through the use of a heterologous signalsequence.

The invention also provides TP-2 chimeric or fusion proteins. As usedherein, a TP-2 “chimeric protein” or “fusion protein” comprises a TP-2polypeptide operatively linked to a non-TP-2 polypeptide. An “TP-2polypeptide” refers to a polypeptide having an amino acid sequencecorresponding to TP-2, whereas a “non-TP-2 polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a polypeptidewhich is not substantially homologous to the TP-2 polypeptide, e.g., apolypeptide which is different from the TP-2 polypeptide and which isderived from the same or a different organism. Within a TP-2 fusionprotein the TP-2 polypeptide can correspond to all or a portion of aTP-2 polypeptide. In a preferred embodiment, a TP-2 fusion proteincomprises at least one biologically active portion of a TP-2polypeptide. In another preferred embodiment, a TP-2 fusion proteincomprises at least two biologically active portions of a TP-2polypeptide. Within the fusion protein, the term “operatively linked” isintended to indicate that the TP-2 polypeptide and the non-TP-2polypeptide are fused in-frame to each other. The non-TP-2 polypeptidecan be fused to the N-terminus or C-terminus of the TP-2 polypeptide.

For example, in one embodiment, the fusion protein is a GST-TP-2 fusionprotein in which the TP-2 sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant TP-2.

In another embodiment, the fusion protein is a TP-2 polypeptidecontaining a heterologous signal sequence at its N-terminus. In certainhost cells (e.g., mammalian host cells), expression and/or secretion ofTP-2 can be increased through the use of a heterologous signal sequence.

The invention also provides PLTR-1 chimeric or fusion proteins. As usedherein, a PLTR-1 “chimeric protein” or “fusion protein” comprises aPLTR-1 polypeptide operatively linked to a non-PLTR-1 polypeptide. A“PLTR-1 polypeptide” refers to a polypeptide having an amino acidsequence corresponding to PLTR-1, whereas a “non-PLTR-1 polypeptide”refers to a polypeptide having an amino acid sequence corresponding to aprotein which is not substantially homologous to the PLTR-1 protein,e.g., a protein which is different from the PLTR-1 protein and which isderived from the same or a different organism. Within a PLTR-1 fusionprotein the PLTR-1 polypeptide can correspond to all or a portion of aPLTR-1 protein. In a preferred embodiment, a PLTR-1 fusion proteincomprises at least one biologically active portion of a PLTR-1 protein.In another preferred embodiment, a PLTR-1 fusion protein comprises atleast two biologically active portions of a PLTR-1 protein. Within thefusion protein, the term “operatively linked” is intended to indicatethat the PLTR-1 polypeptide and the non-PLTR-1 polypeptide are fusedin-frame to each other. The non-PLTR-1 polypeptide can be fused to theN-terminus or C-terminus of the PLTR-1 polypeptide.

For example, in one embodiment, the fusion protein is a GST-PLTR-1fusion protein in which the PLTR-1 sequences are fused to the C-terminusof the GST sequences. Such fusion proteins can facilitate thepurification of recombinant PLTR-1. In another embodiment, the fusionprotein is a PLTR-1 protein containing a heterologous signal sequence atits N-terminus. In certain host cells (e.g., mammalian host cells),expression and/or secretion of PLTR-1 can be increased through use of aheterologous signal sequence.

The invention also provides TFM-2 and/or TFM-3 chimeric or fusionproteins. As used herein, a TFM-2 and/or TFM-3 “chimeric protein” or“fusion protein” comprises a TFM-2 and/or TFM-3 polypeptide operativelylinked to a non-TFM-2 and/or TFM-3 polypeptide. A “TFM-2 polypeptide”and a “TFM-3 polypeptide” refers to a polypeptide having an amino acidsequence corresponding to TFM-2 and TFM-3, respectively, whereas a“non-TFM-2 polypeptide” and a “non-TFM-3 polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a polypeptidewhich is not substantially homologous to the TFM-2 and TFM-3polypeptides, respectively, e.g., a polypeptide which is different fromthe TFM-2 and TFM-3 polypeptide and which is derived from the same or adifferent organism. Within a TFM-2 and/or TFM-3 fusion protein the TFM-2and/or TFM-3 polypeptide can correspond to all or a portion of a TFM-2and/or TFM-3 polypeptide. In a preferred embodiment, a TFM-2 and/orTFM-3 fusion protein comprises at least one biologically active portionof a TFM-2 and/or TFM-3 polypeptide. In another preferred embodiment, aTFM-2 and/or TFM-3 fusion protein comprises at least two biologicallyactive portions of a TFM-2 and/or TFM-3 polypeptide. Within the fusionprotein, the term “operatively linked” is intended to indicate that theTFM-2 and/or TFM-3 polypeptide and the non-TFM-2 and/or TFM-3polypeptide are fused in-frame to each other. The non-TFM-2 and/or TFM-3polypeptide can be fused to the N-terminus or C-terminus of the TFM-2and/or TFM-3 polypeptide.

For example, in one embodiment, the fusion protein is a GST-TFM-2 and/orGST-TFM-3 fusion protein in which the TFM-2 and/or TFM-3 sequences arefused to the C-terminus of the GST sequences. Such fusion proteins canfacilitate the purification of recombinant TFM-2 and/or TFM-3.

In another embodiment, the fusion protein is a TFM-2 and/or TFM-3polypeptide containing a heterologous signal sequence at its N-terminus.In certain host cells (e.g., mammalian host cells), expression and/orsecretion of TFM-2 and/or TFM-3 can be increased through the use of aheterologous signal sequence.

The invention also provides 67118, 67067, and/or 62092 chimeric orfusion proteins. As used herein, a 67118, 67067, and/or 62092 “chimericprotein” or “fusion protein” comprises a 67118, 67067, and/or 62092polypeptide operatively linked to a non-67118, a non-67067, and/or anon-62092 polypeptide. A “67118 polypeptide,” a “67067 polypeptide,” anda “62092 polypeptide” refer to a polypeptide having an amino acidsequence corresponding to 67118, 67067, and 62092, respectively, whereasa “non-67118 polypeptide,” a “non-67067 polypeptide,” and a “non-62092polypeptide” refers to a polypeptide having an amino acid sequencecorresponding to a polypeptide which is not substantially homologous tothe 67118, 67067, and 62092 polypeptides, respectively, e.g., apolypeptide which is different from the 67118, 67067, and 62092polypeptide and which is derived from the same or a different organism.Within a 67118, 67067, and/or 62092 fusion protein the 67118, 67067,and/or 62092 polypeptide can correspond to all or a portion of a 67118,67067, and/or 62092 polypeptide. In a preferred embodiment, a 67118,67067, and/or 62092 fusion protein comprises at least one biologicallyactive portion of a 67118, 67067, and/or 62092 polypeptide. In anotherpreferred embodiment, a 67118, 67067, and/or 62092 fusion proteincomprises at least two biologically active portions of a 67118, 67067,and/or 62092 polypeptide. Within the fusion protein, the term“operatively linked” is intended to indicate that the 67118, 67067,and/or 62092 polypeptide and the non-67118, 67067, and/or 62092polypeptide are fused in-frame to each other. The non-67118, 67067,and/or 62092 polypeptide can be fused to the N-terminus or C-terminus ofthe 67118, 67067, and/or 62092 polypeptide.

For example, in one embodiment, the fusion protein is a GST-67118,GST-67067, or GST-62092 fusion protein in which the 67118, 67067, or62092 sequences are fused to the C-terminus of the GST sequences. Suchfusion proteins can facilitate the purification of recombinant 67118,67067, or 62092.

In another embodiment, the fusion protein is a 67118, 67067, and/or62092 polypeptide containing a heterologous signal sequence at itsN-terminus. In certain host cells (e.g., mammalian host cells),expression and/or secretion of 67118, 67067, and/or 62092 can beincreased through the use of a heterologous signal sequence.

The invention also provides HAAT chimeric or fusion proteins. As usedherein, a HAAT “chimeric protein” or “fusion protein” comprises a HAATpolypeptide operatively linked to a non-HAAT polypeptide. A “HAATpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to HAAT, whereas a “non-HAAT polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homologous to the HAAT protein, e.g., aprotein which is different from the HAAT protein and which is derivedfrom the same or a different organism. Within a HAAT fusion protein theHAAT polypeptide can correspond to all or a portion of a HAAT protein.In a preferred embodiment, a HAAT fusion protein comprises at least onebiologically active portion of a HAAT protein. In another preferredembodiment, a HAAT fusion protein comprises at least two biologicallyactive portions of a HAAT protein. Within the fusion protein, the term“operatively linked” is intended to indicate that the HAAT polypeptideand the non-HAAT polypeptide are fused in-frame to each other. Thenon-HAAT polypeptide can be fused to the N-terminus or C-terminus of theHAAT polypeptide.

For example, in one embodiment, the fusion protein is a GST-HAAT fusionprotein in which the HAAT sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant HAAT. In another embodiment, the fusion protein is a HAATprotein containing a heterologous signal sequence at its N-terminus. Incertain host cells (e.g., mammalian host cells), expression and/orsecretion of HAAT can be increased through use of a heterologous signalsequence.

The invention also provides HST-4 and/or HST-5 chimeric or fusionproteins. As used herein, an HST-4 and/or an HST-5 “chimeric protein” or“fusion protein” comprises an HST-4 and/or an HST-5 polypeptideoperatively linked to a non-HST-4 and/or non-HST-5 polypeptide. An“HST-4 polypeptide” or an “HST-5 polypeptide” refers to a polypeptidehaving an amino acid sequence corresponding to HST-4 and/or HST-5,whereas a “non- HST-4 polypeptide” or a “non- HST-5 polypeptide” refersto a polypeptide having an amino acid sequence corresponding to apolypeptide which is not substantially homologous to the HST-4 and/orthe HST-5 polypeptide, e.g., a polypeptide which is different from theHST-4 and/or the HST-5 polypeptide and which is derived from the same ora different organism. Within an HST-4 and/or an HST-5 fusion protein theHST-4 and/or the HST-5 polypeptide can correspond to all or a portion ofan HST-4 and/or an HST-5 polypeptide. In a preferred embodiment, anHST-4 and/or an HST-5 fusion protein comprises at least one biologicallyactive portion of an HST-4 and/or an HST-5 polypeptide. In anotherpreferred embodiment, an HST-4 and/or an HST-5 fusion protein comprisesat least two biologically active portions of an HST-4 and/or an HST-5polypeptide. Within the fusion protein, the term “operatively linked” isintended to indicate that the HST-4 and/or the HST-5 polypeptide and thenon-HST-4 and/or non-HST-5 polypeptide are fused in-frame to each other.The non-HST-4 and/or the non-HST-5 polypeptide can be fused to theN-terminus or C-terminus of the HST-4 and/or the HST-5 polypeptide.

For example, in one embodiment, the fusion protein is a GST-HST-4 and/ora GST-HST-5 fusion protein in which the HST-4 and/or the HST-5 sequencesare fused to the C-terminus of the GST sequences. Such fusion proteinscan facilitate the purification of recombinant HST-4 and/or HST-5.

In another embodiment, the fusion protein is an HST-4 and/or an HST-5polypeptide containing a heterologous signal sequence at its N-terminus.In certain host cells (e.g., mammalian host cells), expression and/orsecretion of HST-4 and/or HST-5 can be increased through the use of aheterologous signal sequence.

The MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or the HST-5 fusion proteins of the invention can beincorporated into pharmaceutical compositions and administered to asubject in vivo. The MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or the HST-5 fusion proteins can beused to affect the bioavailability of an MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or an HST-5substrate. Use of MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 fusion proteins may be usefultherapeutically for the treatment of disorders caused by, for example,(i) aberrant modification or mutation of a gene encoding an MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or an HST-5 polypeptide; (ii) mis-regulation of the MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or the HST-5 gene; and (iii) aberrant post-translationalmodification of an MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or an HST-5 polypeptide.

Moreover, the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4- and/or the HST-5-fusion proteins of theinvention can be used as immunogens to produce anti-MTP-1, anti-OAT,anti-HST-1, anti-TP-2, anti-PLTR-1, anti-TFM-2, anti-TFM-3, anti-67118,anti-67067, anti-62092, anti- HAAT, anti-HST-4 and/or anti-HST-5antibodies in a subject, to purify MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 ligands andin screening assays to identify molecules which inhibit the interactionof MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067,62092,HAAT, HST-4 and/or HST-5 with an MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118,67067,62092, HAAT, HST-4 and/or an HST-5 substrate.

Preferably, an MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118,67067, 62092, HAAT, HST-4 and/or an HST-5 chimeric or fusion protein ofthe invention is produced by standard recombinant DNA techniques. Forexample, DNA fragments coding for the different polypeptide sequencesare ligated together in-frame in accordance with conventionaltechniques, for example by employing blunt-ended or stagger-endedtermini for ligation, restriction enzyme digestion to provide forappropriate termini, filling-in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining, andenzymatic ligation. In another embodiment, the fusion gene can besynthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Current Protocols in Molecular Biology, eds. Ausubel et al.John Wiley & Sons: 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). An MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118,67067, 62092, HAAT, HST-4- and/or an HST-5-encoding nucleic acid can becloned into such an expression vector such that the fusion moiety islinked in-frame to the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or the HST-5 polypeptide.

The present invention also pertains to variants of the MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or the HST-5 polypeptides which function as either MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 agonists (mimetics) or as HST-4 and/or HST-5 antagonists.Variants of the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or the HST-5 polypeptides can be generatedby mutagenesis, e.g., discrete point mutation or truncation of an MTP-1,an OAT, an HST-1, a TP-2, a PLTR-1, a TFM-2, a TFM-3, a 67118, a 67067,a 62092, a HAAT, an HST-4 and/or an HST-5 polypeptide. An agonist of theMTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or the HST-5 polypeptides can retain substantially thesame, or a subset, of the biological activities of the naturallyoccurring form of an MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or an HST-5 polypeptide. Anantagonist of an MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or an HST-5 polypeptide can inhibit one ormore of the activities of the naturally occurring form of the MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4and/or the HST-5 polypeptide by, for example, competitively modulatingan MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4- and/or an HST-5-mediated activity of an MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or anHST-5 polypeptide. Thus, specific biological effects can be elicited bytreatment with a variant of limited function. In one embodiment,treatment of a subject with a variant having a subset of the biologicalactivities of the naturally occurring form of the polypeptide has fewerside effects in a subject relative to treatment with the naturallyoccurring form of the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or the HST-5 polypeptide.

In one embodiment, variants of an MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or an HST-5polypeptide which function as either MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 agonists(mimetics) or as MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 antagonists can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutants,of an MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or an HST-5 polypeptide for MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5 polypeptide agonist or antagonist activity. In one embodiment, avariegated library of MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5 variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5 variants can be produced by, for example, enzymatically ligating amixture of synthetic oligonucleotides into gene sequences such that adegenerate set of potential MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 sequences isexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g., for phage display) containing the set ofMTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5 sequences therein. There are a variety ofmethods which can be used to produce libraries of potential MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 variants from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be performed in anautomatic DNA synthesizer, and the synthetic gene then ligated into anappropriate expression vector. Use of a degenerate set of genes allowsfor the provision, in one mixture, of all of the sequences encoding thedesired set of potential MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5 sequences. Methods forsynthesizing degenerate oligonucleotides are known in the art (see,e.g., Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu.Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al.(1983) Nucleic Acid Res. 11:477.

In addition, libraries of fragments of an MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or an HST-5polypeptide coding sequence can be used to generate a variegatedpopulation of MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 fragments for screening andsubsequent selection of variants of an MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or an HST-5polypeptide. In one embodiment, a library of coding sequence fragmentscan be generated by treating a double stranded PCR fragment of an MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or an HST-5 coding sequence with a nuclease under conditions whereinnicking occurs only about once per molecule, denaturing the doublestranded DNA, renaturing the DNA to form double stranded DNA which caninclude sense/antisense pairs from different nicked products, removingsingle stranded portions from reformed duplexes by treatment with S1nuclease, and ligating the resulting fragment library into an expressionvector. By this method, an expression library can be derived whichencodes N-terminal, C-terminal and internal fragments of various sizesof the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or the HST-5 polypeptide.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5polypeptides. The most widely used techniques, which are amenable tohigh through-put analysis, for screening large gene libraries typicallyinclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates isolation of the vectorencoding the gene whose product was detected. Recursive ensemblemutagenesis (REM), a new technique which enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 variants (Arkin andYouvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delagrave et al.(1993) Protein Engineering 6(3):327-331).

In one embodiment, cell based assays can be exploited to analyze avariegated MTP-1 library. For example, a library of expression vectorscan be transfected into a cell line, e.g., a neuronal cell line, whichordinarily responds to an MTP-1 ligand in a particular MTP-1ligand-dependent manner. The transfected cells are then contacted withan MTP-1 ligand and the effect of expression of the mutant on, e.g.,membrane excitability of MTP-1 can be detected. Plasmid DNA can then berecovered from the cells which score for inhibition, or alternatively,potentiation of signaling by the MTP-1 ligand, and the individual clonesfurther characterized.

An isolated MTP-1 protein, or a portion or fragment thereof, can be usedas an immunogen to generate antibodies that bind MTP-1 using standardtechniques for polyclonal and monoclonal antibody preparation. Afull-length MTP-1 protein can be used or, alternatively, the inventionprovides antigenic peptide fragments of MTP-1 for use as immunogens. Theantigenic peptide of MTP-1 comprises at least 8 amino acid residues ofthe amino acid sequence shown in SEQ ID NO:2 and encompasses an epitopeof MTP-1 such that an antibody raised against the peptide forms aspecific immune complex with the MTP-1 protein. Preferably, theantigenic peptide comprises at least 10 amino acid residues, morepreferably at least 15 amino acid residues, even more preferably atleast 20 amino acid residues, and most preferably at least 30 amino acidresidues. In a preferred embodiment, portions of the extracellulardomains (e.g., extracellular non-transmembrane domains) in the aminoacid sequence of MTP-1 are used as immunogens (e.g., at about residues40-548, at about residues 612-624, at about residue 675-1006, at aboutresidue 1258-1534, at about residues 1603-1645, and at about residues1749-1931 of SEQ ID NO:2).

Preferred epitopes encompassed by the antigenic peptide are regions ofMTP-1 that are located on the surface of the protein, e.g., hydrophilicregions, as well as regions with high antigenicity.

In one embodiment, cell based assays can be exploited to analyze avariegated OAT library. For example, a library of expression vectors canbe transfected into a cell line, e.g., a liver cell line, whichordinarily responds to OAT in a particular OAT substrate-dependentmanner. The transfected cells are then contacted with an OAT substrateand the effect of the expression of the mutant on signaling by the OATsubstrate can be detected, e.g., by measuring levels of OAT substratetransported into or out of the cells, by measuring gene transcription,by measuring cellular proliferation, and/or by measuring activity ofintracellular signaling pathways. Plasmid DNA can then be recovered fromthe cells which score for inhibition, or alternatively, potentiation ofsignaling by the OAT substrate, and the individual clones furthercharacterized.

An isolated OAT protein, or a portion or fragment thereof, can be usedas an immunogen to generate antibodies that bind OAT using standardtechniques for polyclonal and monoclonal antibody preparation. Afull-length OAT protein can be used or, alternatively, the inventionprovides antigenic peptide fragments of OAT for use as immunogens. Theantigenic peptide of OAT comprises at least 8 amino acid residues of theamino acid sequence shown in SEQ ID NO:5 or 8 and encompasses an epitopeof OAT such that an antibody raised against the peptide forms a specificimmune complex with OAT. Preferably, the antigenic peptide comprises atleast 10 amino acid residues, more preferably at least 15 amino acidresidues, even more preferably at least 20 amino acid residues, and mostpreferably at least 30 amino acid residues.

Preferred epitopes encompassed by the antigenic peptide are regions ofOAT that are located on the surface of the protein, e.g., hydrophilicregions, as well as regions with high antigenicity (see, for example,FIGS. 4 and 5).

In one embodiment, cell based assays can be exploited to analyze avariegated HST-1 library. For example, a library of expression vectorscan be transfected into a cell line, e.g., an endothelial cell line,which ordinarily responds to HST-1 in a particular HST-1substrate-dependent manner. The transfected cells are then contactedwith HST-1 and the effect of expression of the mutant on signaling bythe HST-1 substrate can be detected, e.g., by monitoring intracellularcalcium, IP3, or diacylglycerol concentration, phosphorylation profileof intracellular proteins, or the activity of an HST-1-regulatedtranscription factor. Plasmid DNA can then be recovered from the cellswhich score for inhibition, or alternatively, potentiation of signalingby the HST-1 substrate, and the individual clones further characterized.

An isolated HST-1 polypeptide, or a portion or fragment thereof, can beused as an immunogen to generate antibodies that bind HST-1 usingstandard techniques for polyclonal and monoclonal antibody preparation.A full-length HST-1 polypeptide can be used or, alternatively, theinvention provides antigenic peptide fragments of HST-1 for use asimmunogens. The antigenic peptide of HST-1 comprises at least 8 aminoacid residues of the amino acid sequence shown in SEQ ID NO:13 andencompasses an epitope of HST-1 such that an antibody raised against thepeptide forms a specific immune complex with HST-1. Preferably, theantigenic peptide comprises at least 10 amino acid residues, morepreferably at least 15 amino acid residues, even more preferably atleast 20 amino acid residues, and most preferably at least 30 amino acidresidues.

Preferred epitopes encompassed by the antigenic peptide are regions ofHST-1 that are located on the surface of the polypeptide, e.g.,hydrophilic regions, as well as regions with high antigenicity (see, forexample, FIG. 7).

In one embodiment, cell based assays can be exploited to analyze avariegated TP-2 library. For example, a library of expression vectorscan be transfected into a cell line, e.g., an endothelial cell line,which ordinarily responds to TP-2 in a particular TP-2substrate-dependent manner. The transfected cells are then contactedwith TP-2 and the effect of expression of the mutant on signaling by theTP-2 substrate can be detected, e.g., by monitoring intra-cellularcalcium, IP3, or diacylglycerol concentration, phosphorylation profileof intra-cellular proteins, or the activity of a TP-2-regulatedtranscription factor. Plasmid DNA can then be recovered from the cellswhich score for inhibition, or alternatively, potentiation of signalingby the TP-2 substrate, and the individual clones further characterized.

An isolated TP-2 polypeptide, or a portion or fragment thereof, can beused as an immunogen to generate antibodies that bind TP-2 usingstandard techniques for polyclonal and monoclonal antibody preparation.A full-length TP-2 polypeptide can be used or, alternatively, theinvention provides antigenic peptide fragments of TP-2 for use asimmunogens. The antigenic peptide of TP-2 comprises at least 8 aminoacid residues of the amino acid sequence shown in SEQ ID NO:16 andencompasses an epitope of TP-2 such that an antibody raised against thepeptide forms a specific immune complex with TP-2. Preferably, theantigenic peptide comprises at least 10 amino acid residues, morepreferably at least 15 amino acid residues, even more preferably atleast 20 amino acid residues, and most preferably at least 30 amino acidresidues.

Preferred epitopes encompassed by the antigenic peptide are regions ofTP-2 that are located on the surface of the polypeptide, e.g.,hydrophilic regions, as well as regions with high antigenicity (see, forexample, FIG. 11).

In one embodiment, cell based assays can be exploited to analyze avariegated PLTR-1 library. For example, a library of expression vectorscan be transfected into a cell line which ordinarily responds to PLTR-1in a particular PLTR-1 substrate-dependent manner. The transfected cellsare then contacted with PLTR-1 and the effect of the expression of themutant on signaling by the PLTR-1 substrate can be detected, e.g.,phospholipid transport (e.g., by measuring phospholipid levels insidethe cell or its various cellular compartments, within various cellularmembranes, or in the extracellular medium), hydrolysis of ATP,phosphorylation or dephosphorylation of the HEAT protein, and/or genetranscription. Plasmid DNA can then be recovered from the cells whichscore for inhibition, or alternatively, potentiation of signaling by theHEAT substrate, or which score for increased or decreased levels ofphospholipid transport or ATP hydrolysis, and the individual clonesfurther characterized.

An isolated PLTR-1 protein, or a portion or fragment thereof, can beused as an immunogen to generate antibodies that bind PLTR-1 usingstandard techniques for polyclonal and monoclonal antibody preparation.A full-length PLTR-1 protein can be used or, alternatively, theinvention provides antigenic peptide fragments of PLTR-1 for use asimmunogens. The antigenic peptide of PLTR-1 comprises at least 8 aminoacid residues of the amino acid sequence shown in SEQ ID NO:20 andencompasses an epitope of PLTR-1 such that an antibody raised againstthe peptide forms a specific immune complex with PLTR-1. Preferably, theantigenic peptide comprises at least 10 amino acid residues, morepreferably at least 15 amino acid residues, even more preferably atleast 20 amino acid residues, and most preferably at least 30 amino acidresidues.

Preferred epitopes encompassed by the antigenic peptide are regions ofPLTR-1 that are located on the surface of the protein, e.g., hydrophilicregions, as well as regions with high antigenicity (see, for example,FIG. 15).

In one embodiment, cell based assays can be exploited to analyze avariegated TFM-2 and/or TFM-3 library. For example, a library ofexpression vectors can be transfected into a cell line, e.g., anendothelial cell line, which ordinarily responds to TFM-2 and/or TFM-3in a particular TFM-2 and/or TFM-3 substrate-dependent manner. Thetransfected cells are then contacted with TFM-2 and/or TFM-3 and theeffect of expression of the mutant on signaling by the TFM-2 and/orTFM-3 substrate can be detected, e.g., by monitoring intra-cellularcalcium, IP3, or diacylglycerol concentration, phosphorylation profileof intra-cellular proteins, or the activity of a TFM-2 and/orTFM-3-regulated transcription factor. Plasmid DNA can then be recoveredfrom the cells which score for inhibition, or alternatively,potentiation of signaling by the TFM-2 and/or TFM-3 substrate, and theindividual clones further characterized.

An isolated TFM-2 and/or TFM-3 polypeptide, or a portion or fragmentthereof, can be used as an immunogen to generate antibodies that bindTFM-2 and/or TFM-3 using standard techniques for polyclonal andmonoclonal antibody preparation. A full-length TFM-2 and/or TFM-3polypeptide can be used or, alternatively, the invention providesantigenic peptide fragments of TFM-2 and/or TFM-3 for use as immunogens.The antigenic peptide of TFM-2 and/or TFM-3 comprises at least 8 aminoacid residues of the amino acid sequence shown in SEQ ID NO:28 or 31 andencompasses an epitope of TFM-2 and/or TFM-3 such that an antibodyraised against the peptide forms a specific immune complex with TFM-2and/or TFM-3. Preferably, the antigenic peptide comprises at least 10amino acid residues, more preferably at least 15 amino acid residues,even more preferably at least 20 amino acid residues, and mostpreferably at least 30 amino acid residues.

Preferred epitopes encompassed by the antigenic peptide are regions ofTFM-2 and/or TFM-3 that are located on the surface of the polypeptide,e.g., hydrophilic regions, as well as regions with high antigenicity(see, for example, FIGS. 16 and 18).

In one embodiment, cell based assays can be exploited to analyze avariegated 67118 or 67067 library. For example, a library of expressionvectors can be transfected into a cell line, which ordinarily respondsto 67118 or 67067 in a particular 67118 or 67067 substrate-dependentmanner. The transfected cells are then contacted with 67118 or 67067 andthe effect of the expression of the mutant on signaling by the 67118 or67067 substrate can be detected, e.g., the effect on phospholipidtransport (e.g., by measuring phospholipid levels inside the cell or itsvarious cellular compartments, within various cellular membranes, or inthe extra-cellular medium), hydrolysis of ATP, phosphorylation ordephosphorylation of the HEAT protein, and/or gene transcription.Plasmid DNA can then be recovered from the cells which score forinhibition, or alternatively, potentiation of signaling by the HEATsubstrate, or which score for increased or decreased levels ofphospholipid transport or ATP hydrolysis, and the individual clonesfurther characterized.

In another embodiment, cell based assays can be exploited to analyze avariegated 62092 library. For example, a library of expression vectorscan be transfected into a cell line which ordinarily responds to 62092in a particular 62092 substrate-dependent manner. The transfected cellsare then contacted with 62092 and the effect of the expression of themutant on signaling by the 62092 substrate can be detected, e.g., bymeasuring levels of free or 62092 bound nucleotides, cleavednucleotides, gene transcription, and/or cell proliferation, growth,differentiation, or apoptosis. Plasmid DNA can then be recovered fromthe cells which score for inhibition, or alternatively, potentiation ofsignaling by the 62092 substrate, and the individual clones furthercharacterized.

An isolated 67118, 67067, and/or 62092 polypeptide, or a portion orfragment thereof, can be used as an immunogen to generate antibodiesthat bind 67118, 67067, and/or 62092 using standard techniques forpolyclonal and monoclonal antibody preparation. A full-length 67118,67067, and/or 62092 polypeptide can be used or, alternatively, theinvention provides antigenic peptide fragments of 67118, 67067, and/or62092 for use as immunogens. The antigenic peptide of 67118, 67067,and/or 62092 comprises at least 8 amino acid residues of the amino acidsequence shown in SEQ ID NO:34, 37, or 40 and encompasses an epitope of67118, 67067, and/or 62092 such that an antibody raised against thepeptide forms a specific immune complex with 67118, 67067, and/or 62092.Preferably, the antigenic peptide comprises at least 10 amino acidresidues, more preferably at least 15 amino acid residues, even morepreferably at least 20 amino acid residues, and most preferably at least30 amino acid residues.

Preferred epitopes encompassed by the antigenic peptide are regions of67118, 67067, and/or 62092 that are located on the surface of thepolypeptide, e.g., hydrophilic regions, as well as regions with highantigenicity (see, for example, FIGS. 20, 22, and 24, respectively).

In one embodiment, cell based assays can be exploited to analyze avariegated HAAT library. For example, a library of expression vectorscan be transfected into a cell line which ordinarily responds to HAAT ina particular HAAT substrate-dependent manner. The transfected cells arethen contacted with HAAT and the effect of the expression of the mutanton the HAAT substrate can be detected, e.g., amino acid transport (e.g.,by measuring amino acid levels inside the cell or its various cellularcompartments, within various cellular membranes, or in the extracellularmedium), and/or gene transcription. Plasmid DNA can then be recoveredfrom the cells which score for increased or decreased levels of aminoacid transport, and the individual clones further characterized.

An isolated HAAT protein, or a portion or fragment thereof, can be usedas an immunogen to generate antibodies that bind HAAT using standardtechniques for polyclonal and monoclonal antibody preparation. Afull-length HAAT protein can be used or, alternatively, the inventionprovides antigenic peptide fragments of HAAT for use as immunogens. Theantigenic peptide of HAAT comprises at least 8 amino acid residues ofthe amino acid sequence shown in SEQ ID NO:52 and encompasses an epitopeof HAAT such that an antibody raised against the peptide forms aspecific immune complex with HAAT. Preferably, the antigenic peptidecomprises at least 10 amino acid residues, more preferably at least 15amino acid residues, even more preferably at least 20 amino acidresidues, and most preferably at least 30 amino acid residues.

Preferred epitopes encompassed by the antigenic peptide are regions ofHAAT that are located on the surface of the protein, e.g., hydrophilicregions, as well as regions with high antigenicity (see, for example,FIG. 26).

In one embodiment, cell based assays can be exploited to analyze avariegated HST-4 and/or HST-5 library. For example, a library ofexpression vectors can be transfected into a cell line, e.g., anendothelial cell line, which ordinarily responds to HST-4 and/or HST-5in a particular HST-4 and/or HST-5 substrate-dependent manner. Thetransfected cells are then contacted with HST-4 and/or HST-5 and theeffect of expression of the mutant on signaling by the HST-4 and/or theHST-5 substrate can be detected, e.g., by monitoring intracellularcalcium, IP3, or diacylglycerol concentration, phosphorylation profileof intracellular proteins, or the activity of an HST-4- and/or anHST-5-regulated transcription factor. Plasmid DNA can then be recoveredfrom the cells which score for inhibition, or alternatively,potentiation of signaling by the HST-4 and/or the HST-5 substrate, andthe individual clones further characterized.

An isolated HST-4 and/or HST-5 polypeptide, or a portion or fragmentthereof, can be used as an immunogen to generate antibodies that bindHST-4 and/or HST-5 using standard techniques for polyclonal andmonoclonal antibody preparation. A full-length HST-4 and/or HST-5polypeptide can be used or, alternatively, the invention providesantigenic peptide fragments of HST-4 and/or HST-5 for use as immunogens.The antigenic peptide of HST-4 and/or HST-5 comprises at least 8 aminoacid residues of the amino acid sequence shown in SEQ ID NO:55 or 58 andencompasses an epitope of HST-4 and/or HST-5 such that an antibodyraised against the peptide forms a specific immune complex with HST-4and/or HST-5. Preferably, the antigenic peptide comprises at least 10amino acid residues, more preferably at least 15 amino acid residues,even more preferably at least 20 amino acid residues, and mostpreferably at least 30 amino acid residues.

Preferred epitopes encompassed by the antigenic peptide are regions ofHST-4 and/or HST-5 that are located on the surface of the polypeptide,e.g., hydrophilic regions, as well as regions with high antigenicity(see, for example, FIGS. 29 and 30).

An MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or an HST-5 immunogen typically is used to prepareantibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouseor other mammal) with the immunogen. An appropriate immunogenicpreparation can contain, for example, recombinantly expressed MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 polypeptide or a chemically synthesized MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5 polypeptide. The preparation can further include an adjuvant, suchas Freund's complete or incomplete adjuvant, or similarimmunostimulatory agent. Immunization of a suitable subject with animmunogenic HST-4 and/or HST-5 preparation induces a polyclonalanti-MTP-1, anti-OAT, anti-HST-1, anti-TP-2, anti-PLTR-1, anti-TFM-2,anti-TFM-3, anti-67118, anti-67067, anti-62092, anti- HAAT, anti-HST-4and/or anti-HST-5 antibody response.

Accordingly, another aspect of the invention pertains to anti-MTP-1,anti-OAT, anti-HST-1, anti-TP-2, anti-PLTR-1, anti-TFM-2, anti-TFM-3,anti-67118, anti-67067, anti-62092, anti- HAAT, anti-HST-4 and/oranti-HST-5 antibodies. The term “antibody” as used herein refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) an antigen,such as MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5. Examples of immunologically activeportions of immunoglobulin molecules include F(ab) and F(ab′)₂ fragmentswhich can be generated by treating the antibody with an enzyme such aspepsin. The invention provides polyclonal and monoclonal antibodies thatbind MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5. The term “monoclonal antibody” or “monoclonalantibody composition”, as used herein, refers to a population ofantibody molecules that contain only one species of an antigen bindingsite capable of immunoreacting with a particular epitope of MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5. A monoclonal antibody composition thus typically displaysa single binding affinity for a particular MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5polypeptide with which it immunoreacts.

Polyclonal anti-MTP-1, anti-OAT, anti-HST-1, anti-TP-2, anti-PLTR-1,anti-TFM-2, anti-TFM-3, anti-67118, anti-67067, anti-62092, anti- HAAT,anti-HST-4 and/or anti-HST-5 antibodies can be prepared as describedabove by immunizing a suitable subject with an MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or an HST-5immunogen. The anti-MTP-1, anti-OAT, anti-HST-1, anti-TP-2, anti-PLTR-1,anti-TFM-2, anti-TFM-3, anti-67118, anti-67067, anti-62092, anti- HAAT,anti-HST-4 and/or anti-HST-5 antibody titer in the immunized subject canbe monitored over time by standard techniques, such as with an enzymelinked immunosorbent assay (ELISA) using immobilized MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5. If desired, the antibody molecules directed against MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 can be isolated from the mammal (e.g., from the blood) andfurther purified by well known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the anti-MTP-1, anti-OAT, anti-HST-1,anti-TP-2, anti-PLTR-1, anti-TFM-2, anti-TFM-3, anti-67118, anti-67067,anti-62092, anti-HAAT, anti-HST-4 and/or anti-HST-5 antibody titers arehighest, antibody-producing cells can be obtained from the subject andused to prepare monoclonal antibodies by standard techniques, such asthe hybridoma technique originally described by Kohler and Milstein(1975) Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol.127:539-46; Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al.(1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int.J. Cancer 29:269-75), the more recent human B cell hybridoma technique(Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridoma technique(Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96) or trioma techniques. The technology forproducing monoclonal antibody hybridomas is well known (see generally R.H. Kenneth, in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, New York (1980); E. A.Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977)Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typicallya myeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with an MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or an HST-5 immunogen as described above,and the culture supernatants of the resulting hybridoma cells arescreened to identify a hybridoma producing a monoclonal antibody thatbinds MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-MTP-1, anti-OAT, anti-HST-1, anti-TP-2, anti-PLTR-1, anti-TFM-2,anti-TFM-3, anti-67118, anti-67067, anti-62092, anti- HAAT, anti-HST-4and/or anti-HST-5 monoclonal antibody (see, e.g., G. Galfre et al.(1977) Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra;Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies,cited supra). Moreover, the ordinarily skilled worker will appreciatethat there are many variations of such methods which also would beuseful. Typically, the immortal cell line (e.g., a myeloma cell line) isderived from the same mammalian species as the lymphocytes. For example,murine hybridomas can be made by fusing lymphocytes from a mouseimmunized with an immunogenic preparation of the present invention withan immortalized mouse cell line. Preferred immortal cell lines are mousemyeloma cell lines that are sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a numberof myeloma cell lines can be used as a fusion partner according tostandard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC(American Type Culture Collection, Manassas, Va.). Typically,HAT-sensitive mouse myeloma cells are fused to mouse splenocytes usingpolyethylene glycol (“PEG”). Hybridoma cells resulting from the fusionare then selected using HAT medium, which kills unfused andunproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindMTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-MTP-1, anti-OAT, anti-HST-1, anti-TP-2,anti-PLTR-1,anti- TFM-2, anti-TFM-3, anti-67118, anti-67067, anti-62092,anti- HAAT, anti-HST-4 and/or anti-HST-5 antibody can be identified andisolated by screening a recombinant combinatorial immunoglobulin library(e.g., an antibody phage display library) with MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 tothereby isolate immunoglobulin library members that bind MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5. Kits for generating and screening phage display librariesare commercially available (e.g., the Pharmacia Recombinant PhageAntibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP™Phage Display Kit, Catalog No. 240612). Additionally, examples ofmethods and reagents particularly amenable for use in generating andscreening antibody display library can be found in, for example, Ladneret al. U.S. Pat. No. 5,223,409; Kang et al. PCT InternationalPublication No. WO 92/18619; Dower et al. PCT International PublicationNo. WO 91/17271; Winter et al. PCT International Publication WO92/20791; Markland et al. PCT International Publication No. WO 92/15679;Breitling et al. PCT International Publication WO 93/01288; McCaffertyet al. PCT International Publication No. WO 92/01047; Garrard et al. PCTInternational Publication No. WO 92/09690; Ladner et al. PCTInternational Publication No. WO 90/02809; Fuchs et al. (1991)Bio/Technology 9:1369-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol. Biol.226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrard et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc. Acid Res.19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

Additionally, recombinant anti-MTP-1, anti-OAT, anti-HST-1, anti-TP-2,anti-PLTR-1,anti- TFM-2, anti-TFM-3, anti-67118, anti-67067, anti-62092,anti- HAAT, anti-HST-4 and/or anti-HST-5 antibodies, such as chimericand humanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.International Application No. PCT/US86/02269; Akira, et al. EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT International Publication No. WO 86/01533; Cabilly et al.U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987)Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol.139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218;Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al. (1985)Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyen et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

An anti-MTP-1, anti-OAT, anti-HST-1, anti-TP-2, anti-PLTR-1, anti-TFM-2, anti-TFM-3, anti-67118, anti-67067, anti-62092, anti- HAAT,anti-HST-4 and/or anti-HST-5 antibody (e.g., monoclonal antibody) can beused to isolate MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 by standard techniques, such asaffinity chromatography or immunoprecipitation. An anti-MTP-1, anti-OAT,anti-HST-1, anti-TP-2, anti-PLTR-1, anti- TFM-2, anti-TFM-3, anti-67118,anti-67067, anti-62092, anti-HAAT, anti-HST-4 and/or anti-HST-5 antibodycan facilitate the purification of natural MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 fromcells and of recombinantly produced MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 expressed inhost cells. Moreover, an anti-MTP-1, anti-OAT, anti-HST-1, anti-TP-2,anti-PLTR-1,anti- TFM-2, anti-TFM-3, anti-67118, anti-67067, anti-62092,anti-HAAT, anti-HST-4 and/or anti-HST-5 antibody can be used to detectMTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5 polypeptides (e.g., in a cellular lysate orcell supernatant) in order to evaluate the abundance and pattern ofexpression of the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 polypeptides. Anti-MTP-1,anti-OAT, anti-HST-1, anti-TP-2, anti-PLTR-1,anti- TFM-2, anti-TFM-3,anti-67118, anti-67067, anti-62092, anti-HAAT, anti-HST-4 and/oranti-HST-5 antibodies can be used diagnostically to monitor polypeptidelevels in tissue as part of a clinical testing procedure, e.g., to, forexample, determine the efficacy of a given treatment regimen. Detectioncan be facilitated by coupling (i.e., physically linking) the antibodyto a detectable substance. Examples of detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding an MTP-1 protein(or a portion thereof). Another aspect of the invention pertains tovectors, for example recombinant expression vectors, containing an OATnucleic acid molecule or vectors containing a nucleic acid moleculewhich encodes an OAT protein (or a portion thereof). Another aspect ofthe invention pertains to vectors, for example recombinant expressionvectors, containing a nucleic acid containing an HST-1 nucleic acidmolecule or vectors containing a nucleic acid molecule which encodes anHST-1 polypeptide (or a portion thereof). Another aspect of theinvention pertains to vectors, for example recombinant expressionvectors, containing a nucleic acid containing a TP-2 nucleic acidmolecule or vectors containing a nucleic acid molecule which encodes aTP-2 polypeptide (or a portion thereof). Another aspect of the inventionpertains to vectors, for example recombinant expression vectors,containing a PLTR-1 nucleic acid molecule or vectors containing anucleic acid molecule which encodes a PLTR-1 protein (or a portionthereof). Another aspect of the invention pertains to vectors, forexample recombinant expression vectors, containing a nucleic acidcontaining a TFM-2 and/or TFM-3 nucleic acid molecule or vectorscontaining a nucleic acid molecule which encodes a TFM-2 and/or TFM-3polypeptide (or a portion thereof). Another aspect of the inventionpertains to vectors, for example recombinant expression vectors,containing a nucleic acid containing a 67118, 67067, and/or 62092nucleic acid molecule or vectors containing a nucleic acid moleculewhich encodes a 67118, 67067, and/or 62092 polypeptide (or a portionthereof). Another aspect of the invention pertains to vectors, forexample recombinant expression vectors, containing a HAAT nucleic acidmolecule or vectors containing a nucleic acid molecule which encodes aHAAT protein (or a portion thereof). Another aspect of the inventionpertains to vectors, for example recombinant expression vectors,containing a nucleic acid containing an HST-4 and/or an HST-5 nucleicacid molecule or vectors containing a nucleic acid molecule whichencodes an HST-4 and/or an HST-5 polypeptide (or a portion thereof). Asused herein, the term “vector” refers to a nucleic acid molecule capableof transporting another nucleic acid to which it has been linked. Onetype of vector is a “plasmid”, which refers to a circular doublestranded DNA loop into which additional DNA segments can be ligated.Another type of vector is a viral vector, wherein additional DNAsegments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cells and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of polypeptide desired, and the like. The expressionvectors of the invention can be introduced into host cells to therebyproduce proteins or peptides, including fusion proteins or peptides,encoded by nucleic acids as described herein (e.g., MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5 polypeptides, mutant forms of MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5polypeptides, fusion proteins, and the like).

Accordingly, an exemplary embodiment provides a method for producing aprotein, preferably an OAT protein, by culturing in a suitable medium ahost cell of the invention (e.g., a mammalian host cell such as anon-human mammalian cell) containing a recombinant expression vector,such that the protein is produced.

Accordingly, an exemplary embodiment provides a method for producing apolypeptide, preferably an HST-1 polypeptide, by culturing in a suitablemedium a host cell of the invention (e.g., a mammalian host cell such asa non-human mammalian cell) containing a recombinant expression vector,such that the polypeptide is produced.

Accordingly, an exemplary embodiment provides a method for producing apolypeptide, preferably a TP-2 polypeptide, by culturing in a suitablemedium a host cell of the invention (e.g., a mammalian host cell such asa non-human mammalian cell) containing a recombinant expression vector,such that the polypeptide is produced.

Accordingly, an exemplary embodiment provides a method for producing aprotein, preferably a PLTR-l protein, by culturing in a suitable mediuma host cell of the invention (e.g., a mammalian host cell such as anon-human mammalian cell) containing a recombinant expression vector,such that the protein is produced.

Accordingly, an exemplary embodiment provides a method for producing apolypeptide, preferably a TFM-2 and/or TFM-3 polypeptide, by culturingin a suitable medium a host cell of the invention (e.g., a mammalianhost cell such as a non-human mammalian cell) containing a recombinantexpression vector, such that the polypeptide is produced.

Accordingly, an exemplary embodiment provides a method for producing apolypeptide, preferably a 67118, 67067, and/or 62092 polypeptide, byculturing in a suitable medium a host cell of the invention (e.g., amammalian host cell such as a non-human mammalian cell) containing arecombinant expression vector, such that the polypeptide is produced.

Accordingly, an exemplary embodiment provides a method for producing aprotein, preferably a HAAT protein, by culturing in a suitable medium ahost cell of the invention (e.g., a mammalian host cell such as anon-human mammalian cell) containing a recombinant expression vector,such that the protein is produced.

Accordingly, an exemplary embodiment provides a method for producing apolypeptide, preferably an HST-4 and/or an HST-5 polypeptide, byculturing in a suitable medium a host cell of the invention (e.g., amammalian host cell such as a non-human mammalian cell) containing arecombinant expression vector, such that the polypeptide is produced.

The recombinant expression vectors of the invention can be designed forexpression of MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 polypeptides in prokaryotic oreukaryotic cells. For example, MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 polypeptides can beexpressed in bacterial cells such as E. coli, insect cells (usingbaculovirus expression vectors) yeast cells or mammalian cells. Suitablehost cells are discussed further in Goeddel, Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

Purified fusion proteins can be utilized in MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5activity assays, (e.g., direct assays or competitive assays described indetail below), or to generate antibodies specific for MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5 polypeptides, for example. In a preferred embodiment, an MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 fusion protein expressed in a retroviral expression vectorof the present invention can be utilized to infect bone marrow cellswhich are subsequently transplanted into irradiated recipients. Thepathology of the subject recipient is then examined after sufficienttime has passed (e.g., six (6) weeks).

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a residentprophage harboring a T7 gn1 gene under the. transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or the HST-5 expressionvector is a yeast expression vector. Examples of vectors for expressionin yeast S. cerevisiae include pYepSec1 (Baldari, et al., (1987) EMBO J.6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88(Schultz et al., (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation,San Diego, Calif.), and picZ (Invitrogen Corporation, San Diego,Calif.).

Alternatively, MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 polypeptides can be expressed ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5 mRNA. Regulatory sequencesoperatively linked to a nucleic acid cloned in the antisense orientationcan be chosen which direct the continuous expression of the antisenseRNA molecule in a variety of cell types, for instance viral promotersand/or enhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal., Antisense RNA as a molecular tool for genetic analysis,Reviews—Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which anMTP-1, an OAT, an HST-1, a TP-2, a PLTR-1, a TFM-2, a TFM-3, a 67118, a67067, a 62092, a HAAT, an HST-4 and/or an HST-5 nucleic acid moleculeof the invention is introduced, e.g., an MTP-1, an OAT, an HST-1, aTP-2, a PLTR-1, a TFM-2, a TFM-3, a 67118, a 67067, a 62092, a HAAT, anHST-4 and/or an HST-5 nucleic acid molecule within a vector (e.g., arecombinant expression vector) or an MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or an HST-5 nucleicacid molecule containing sequences which allow it to homologouslyrecombine into a specific site of the host cell's genome. The terms“host cell” and “recombinant host cell” are used interchangeably herein.It is understood that such terms refer not only to the particularsubject cell but to the progeny or potential progeny of such a cell.Because certain modifications may occur in succeeding generations due toeither mutation or environmental influences, such progeny may not, infact, be identical to the parent cell, but are still included within thescope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, anMTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or an HST-5 polypeptide can be expressed in bacterialcells such as E. coli, insect cells, yeast or mammalian cells (such asChinese hamster ovary cells (CHO) or COS cells). Other suitable hostcells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding an MTP-1, an OAT, an HST-1, a TP-2, a PLTR-1, aTFM-2, a TFM-3, a 67118, a 67067, a 62092, a HAAT, an HST-4 and/or anHST-5 polypeptide or can be introduced on a separate vector. Cellsstably transfected with the introduced nucleic acid can be identified bydrug selection (e.g., cells that have incorporated the selectable markergene will survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) an MTP-1, anOAT, an HST-1, a TP-2, a PLTR-1, a TFM-2, a TFM-3, a 67118, a 67067, a62092, a HAAT, an HST-4 and/or an HST-5 polypeptide. Accordingly, theinvention further provides methods for producing an MTP-1, an OAT, anHST-1, a TP-2, a PLTR-1, a TFM-2, a TFM-3, a 67118, a 67067, a 62092, aHAAT, an HST-4 and/or an HST-5 polypeptide using the host cells of theinvention. In one embodiment, the method comprises culturing the hostcell of the invention (into which a recombinant expression vectorencoding an MTP-1, an OAT, an HST-1, a TP-2, a PLTR-1, a TFM-2, a TFM-3,a 67118, a 67067, a 62092, a HAAT, an HST-4 and/or an HST-5 polypeptidehas been introduced) in a suitable medium such that an MTP-1, an OAT, anHST-1, a TP-2, a PLTR-1, a TFM-2, a TFM-3, a 67118, a 67067, a 62092, aHAAT, an HST-4 and/or an HST-5 polypeptide is produced. In anotherembodiment, the method further comprises isolating an MTP-1, an OAT, anHST-1, a TP-2, a PLTR-1, a TFM-2, a TFM-3, a 67118, a 67067, a 62092, aHAAT, an HST-4 and/or an HST-5 polypeptide from the medium or the hostcell.

The host cells of the invention can also be used to produce non-humantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichMTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4- and/or HST-5-coding sequences have been introduced. Suchhost cells can then be used to create non-human transgenic animals inwhich exogenous HST-4 and/or HST-5 sequences have been introduced intotheir genome or homologous recombinant animals in which endogenousMTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5 sequences have been altered. Such animals areuseful for studying the function and/or activity of an MTP-1, an OAT, anHST-1, a TP-2, a PLTR-1, a TFM-2, a TFM-3, a 67118, a 67067, a 62092, aHAAT, an HST-4 and/or an HST-5 and for identifying and/or evaluatingmodulators of MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 activity. As used herein, a“transgenic animal” is a non-human animal, preferably a mammal, morepreferably a rodent such as a rat or mouse, in which one or more of thecells of the animal includes a transgene. Other examples of transgenicanimals include non-human primates, sheep, dogs, cows, goats, chickens,amphibians, and the like. A transgene is exogenous DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops and which remains in the genome of the mature animal, therebydirecting the expression of an encoded gene product in one or more celltypes or tissues of the transgenic animal. As used herein, a “homologousrecombinant animal” is a non-human animal, preferably a mammal, morepreferably a mouse, in which an endogenous MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 genehas been altered by homologous recombination between the endogenous geneand an exogenous DNA molecule introduced into a cell of the animal,e.g., an embryonic cell of the animal, prior to development of theanimal.

A transgenic animal of the invention can be created by introducing anMTP-1-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The MTP-1cDNA sequence of SEQ ID NO:1 can be introduced as a transgene into thegenome of a non-human animal. Alternatively, a nonhuman homologue of ahuman MTP-1 gene, such as a mouse or rat MTP-1 gene, can be used as atransgene. Alternatively, an MTP-1 gene homologue, such as another MTP-1family member, can be isolated based on hybridization to the MTP-1 cDNAsequences of SEQ ID NO:1 or 3, and used as a transgene. Intronicsequences and polyadenylation signals can also be included in thetransgene to increase the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to anMTP-1 transgene to direct expression of an MTP-1 protein to particularcells. Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of an MTP-1 transgene in its genome and/or expression of MTP-1mRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene encoding an MTP-1protein can further be bred to other transgenic animals carrying othertransgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of an MTP-1 gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the MTP-1 gene. The MTP-1 gene can be a human gene(e.g., the cDNA of SEQ ID NO:3), but more preferably, is a non-humanhomologue of a human MTP-1 gene (e.g., a cDNA isolated by stringenthybridization with the nucleotide sequence of SEQ ID NO:1). For example,a mouse MTP-1 gene can be used to construct a homologous recombinationnucleic acid molecule, e.g., a vector, suitable for altering anendogenous MTP-1 gene in the mouse genome. In a preferred embodiment,the homologous recombination nucleic acid molecule is designed suchthat, upon homologous recombination, the endogenous MTP-1 gene isfunctionally disrupted (i.e., no longer encodes a functional protein;also referred to as a “knock out” vector). Alternatively, the homologousrecombination nucleic acid molecule can be designed such that, uponhomologous recombination, the endogenous MTP-1 gene is mutated orotherwise altered but still encodes functional protein (e.g., theupstream regulatory region can be altered to thereby alter theexpression of the endogenous MTP-1 protein). In the homologousrecombination nucleic acid molecule, the altered portion of the MTP-1gene is flanked at its 5′ and 3′ ends by additional nucleic acidsequence of the MTP-1 gene to allow for homologous recombination tooccur between the exogenous MTP-1 gene carried by the homologousrecombination nucleic acid molecule and an endogenous MTP-1 gene in acell, e.g., an embryonic stem cell. The additional flanking MTP-1nucleic acid sequence is of sufficient length for successful homologousrecombination with the endogenous gene. Typically, several kilobases offlanking DNA (both at the 5′ and 3′ ends) are included in the homologousrecombination nucleic acid molecule (see, e.g., Thomas, K. R. andCapecchi, M. R. (1987) Cell 51:503 for a description of homologousrecombination vectors). The homologous recombination nucleic acidmolecule is introduced into a cell, e.g., an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced MTP-1 genehas homologously recombined with the endogenous MTP-1 gene are selected(see e.g., Li, E. et al. (1992) Cell 69:915). The selected cells canthen injected into a blastocyst of an animal (e.g., a mouse) to formaggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted intoa suitable pseudopregnant female foster animal and the embryo brought toterm. Progeny harboring the homologously recombined DNA in their germcells can be used to breed animals in which all cells of the animalcontain the homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination nucleicacid molecules, e.g., vectors, or homologous recombinant animals aredescribed further in Bradley, A. (1991) Current Opinion in Biotechnology2:823-829 and in PCT International Publication Nos.: WO 90/11354 by LeMouellec et al.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstraet al.; and WO 93/04169 by Berns et al.

A transgenic animal of the invention can be created by introducing anOAT-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection or retroviral infection, and allowingthe oocyte to develop in a pseudopregnant female foster animal. The OATcDNA sequence of SEQ ID NO:4 or 7 can be introduced as a transgene intothe genome of a non-human animal. Alternatively, a non-human homologueof a human OAT gene, such as a rat or mouse OAT gene, can be used as atransgene. Alternatively, an OAT gene homologue, such as another OATfamily member, can be isolated based on hybridization to the OAT cDNAsequences of SEQ ID NO:4, 6, 7, or 9, (described further in subsection Iabove) and used as a transgene. Intronic sequences and polyadenylationsignals can also be included in the transgene to increase the efficiencyof expression of the transgene. A tissue-specific regulatory sequence(s)can be operably linked to an OAT transgene to direct expression of anOAT protein to particular cells. Methods for generating transgenicanimals via embryo manipulation and microinjection, particularly animalssuch as mice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder etal., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1986). Similar methods are used for production ofother transgenic animals. A transgenic founder animal can be identifiedbased upon the presence of an OAT transgene in its genome and/orexpression of OAT mRNA in tissues or cells of the animals. A transgenicfounder animal can then be used to breed additional animals carrying thetransgene. Moreover, transgenic animals carrying a transgene encoding anOAT protein can further be bred to other transgenic animals carryingother transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of an OAT gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the OAT gene. The OAT gene can be a human gene(e.g., the cDNA of SEQ ID NO:4 or 7), but more preferably, is anon-human homologue of a human OAT gene (e.g., a cDNA isolated bystringent hybridization with the nucleotide sequence of SEQ ID NO:4, 6,7, or 9), For example, a mouse OAT gene can be used to construct ahomologous recombination nucleic acid molecule, e.g., a vector, suitablefor altering an endogenous OAT gene in the mouse genome. In a preferredembodiment, the homologous recombination nucleic acid molecule isdesigned such that, upon homologous recombination, the endogenous OATgene is functionally disrupted (i.e., no longer encodes a functionalprotein; also referred to as a “knock out” vector). Alternatively, thehomologous recombination nucleic acid molecule can be designed suchthat, upon homologous recombination, the endogenous OAT gene is mutatedor otherwise altered but still encodes functional protein (e.g., theupstream regulatory region can be altered to thereby alter theexpression of the endogenous OAT protein). In the homologousrecombination nucleic acid molecule, the altered portion of the OAT geneis flanked at its 5′ and 3′ ends by additional nucleic acid sequence ofthe OAT gene to allow for homologous recombination to occur between theexogenous OAT gene carried by the homologous recombination nucleic acidmolecule and an endogenous OAT gene in a cell, e.g., an embryonic stemcell. The additional flanking OAT nucleic acid sequence is of sufficientlength for successful homologous recombination with the endogenous gene.Typically, several kilobases of flanking DNA (both at the 5′ and 3′ends) are included in the homologous recombination nucleic acid molecule(see, e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for adescription of homologous recombination vectors). The homologousrecombination nucleic acid molecule is introduced into a cell, e.g., anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced OAT gene has homologously recombined with the endogenousOAT gene are selected (see e.g., Li, E. et al. (1992) Cell 69:915). Theselected cells can then be injected into a blastocyst of an animal(e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach,Robertson, E. J. ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryocan then be implanted into a suitable pseudopregnant female fosteranimal and the embryo brought to term. Progeny harboring thehomologously recombined DNA in their germ cells can be used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA by germline transmission of the transgene. Methods forconstructing homologous recombination nucleic acid molecules, e.g.,vectors, or homologous recombinant animals are described further inBradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and in PCTInternational Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO93/04169 by Berns et al.

A transgenic animal of the invention can be created by introducing anHST-1-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The HST-1cDNA sequence of SEQ ID NO:12 can be introduced as a transgene into thegenome of a non-human animal. Alternatively, a nonhuman homologue of ahuman HST-1 gene, such as a mouse or rat HST-1 gene, can be used as atransgene. Alternatively, an HST-1 gene homologue, such as another HST-1family member, can be isolated based on hybridization to the HST-1 cDNAsequences of SEQ ID NO:12 or 14 (described further in subsection Iabove) and used as a transgene. Intronic sequences and polyadenylationsignals can also be included in the transgene to increase the efficiencyof expression of the transgene. A tissue-specific regulatory sequence(s)can be operably linked to an HST-1 transgene to direct expression of anHST-1 polypeptide to particular cells. Methods for generating transgenicanimals via embryo manipulation and microinjection, particularly animalssuch as mice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder etal., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986). Similar methods are used for productionof other transgenic animals. A transgenic founder animal can beidentified based upon the presence of an HST-1 transgene in its genomeand/or expression of HST-1 mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding an HST-1 polypeptide can further be bred to othertransgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of an HST-1 gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the HST-1 gene. The HST-1 gene can be a human gene(e.g., the cDNA of SEQ ID NO:14), but more preferably, is a non-humanhomologue of a human HST-1 gene (e.g., a cDNA isolated by stringenthybridization with the nucleotide sequence of SEQ ID NO:12). Forexample, a mouse HST-1 gene can be used to construct a homologousrecombination nucleic acid molecule, e.g., a vector, suitable foraltering an endogenous HST-1 gene in the mouse genome. In a preferredembodiment, the homologous recombination nucleic acid molecule isdesigned such that, upon homologous recombination, the endogenous HST-1gene is functionally disrupted (i.e., no longer encodes a functionalprotein; also referred to as a “knock out” vector). Alternatively, thehomologous recombination nucleic acid molecule can be designed suchthat, upon homologous recombination, the endogenous HST-1 gene ismutated or otherwise altered but still encodes functional polypeptide(e.g., the upstream regulatory region can be altered to thereby alterthe expression of the endogenous HST-1 polypeptide). In the homologousrecombination nucleic acid molecule, the altered portion of the HST-1gene is flanked at its 5′ and 3′ ends by additional nucleic acidsequence of the HST-1 gene to allow for homologous recombination tooccur between the exogenous HST-1 gene carried by the homologousrecombination nucleic acid molecule and an endogenous HST-1 gene in acell, e.g., an embryonic stem cell. The additional flanking HST-1nucleic acid sequence is of sufficient length for successful homologousrecombination with the endogenous gene. Typically, several kilobases offlanking DNA (both at the 5′ and 3′ ends) are included in the homologousrecombination nucleic acid molecule (see, e.g., Thomas, K. R. andCapecchi, M. R. (1987) Cell 51:503 for a description of homologousrecombination vectors). The homologous recombination nucleic acidmolecule is introduced into a cell, e.g., an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced HST-1 genehas homologously recombined with the endogenous HST-1 gene are selected(see e.g., Li, E. et al. (1992) Cell 69:915). The selected cells canthen injected into a blastocyst of an animal (e.g., a mouse) to formaggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted intoa suitable pseudopregnant female foster animal and the embryo brought toterm. Progeny harboring the homologously recombined DNA in their germcells can be used to breed animals in which all cells of the animalcontain the homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination nucleicacid molecules, e.g., vectors, or homologous recombinant animals aredescribed further in Bradley, A. (1991) Current Opinion in Biotechnology2:823-829 and in PCT International Publication Nos.: WO 90/11354 by LeMouellec et al.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijistraet al.; and WO 93/04169 by Berns et al.

A transgenic animal of the invention can be created by introducing aTP-2-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The TP-2cDNA sequence of SEQ ID NO:15 can be introduced as a transgene into thegenome of a non-human animal. Alternatively, a nonhuman homologue of ahuman TP-2 gene, such as a mouse or rat TP-2 gene, can be used as atransgene. Alternatively, a TP-2 gene homologue, such as another TP-2family member, can be isolated based on hybridization to the TP-2 cDNAsequences of SEQ ID NO:15 or 17, (described further in subsection Iabove) and used as a transgene. Intronic sequences and polyadenylationsignals can also be included in the transgene to increase the efficiencyof expression of the transgene. A tissue-specific regulatory sequence(s)can be operably linked to a TP-2 transgene to direct expression of aTP-2 polypeptide to particular cells. Methods for generating transgenicanimals via embryo manipulation and microinjection, particularly animalssuch as mice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder etal., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986). Similar methods are used for productionof other transgenic animals. A transgenic founder animal can beidentified based upon the presence of a TP-2 transgene in its genomeand/or expression of TP-2 mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding a TP-2 polypeptide can further be bred to othertransgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a TP-2 gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the TP-2 gene. The TP-2 gene can be a human gene(e.g., the cDNA of SEQ ID NO:17), but more preferably, is a non-humanhomologue of a human TP-2 gene (e.g., a cDNA isolated by stringenthybridization with the nucleotide sequence of SEQ ID NO:15). Forexample, a mouse TP-2 gene can be used to construct a homologousrecombination nucleic acid molecule, e.g., a vector, suitable foraltering an endogenous TP-2 gene in the mouse genome. In a preferredembodiment, the homologous recombination nucleic acid molecule isdesigned such that, upon homologous recombination, the endogenous TP-2gene is functionally disrupted (i.e., no longer encodes a functionalprotein; also referred to as a “knock out” vector). Alternatively, thehomologous recombination nucleic acid molecule can be designed suchthat, upon homologous recombination, the endogenous TP-2 gene is mutatedor otherwise altered but still encodes functional polypeptide (e.g., theupstream regulatory region can be altered to thereby alter theexpression of the endogenous TP-2 polypeptide). In the homologousrecombination nucleic acid molecule, the altered portion of the TP-2gene is flanked at its 5′ and 3′ ends by additional nucleic acidsequence of the TP-2 gene to allow for homologous recombination to occurbetween the exogenous TP-2 gene carried by the homologous recombinationnucleic acid molecule and an endogenous TP-2 gene in a cell, e.g., anembryonic stem cell. The additional flanking TP-2 nucleic acid sequenceis of sufficient length for successful homologous recombination with theendogenous gene. Typically, several kilobases of flanking DNA (both atthe 5′ and 3′ ends) are included in the homologous recombination nucleicacid molecule (see, e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell51:503 for a description of homologous recombination vectors). Thehomologous recombination nucleic acid molecule is introduced into acell, e.g., an embryonic stem cell line (e.g., by electroporation) andcells in which the introduced TP-2 gene has homologously recombined withthe endogenous TP-2 gene are selected (see e.g., Li, E. et al. (1992)Cell 69:915). The selected cells can then injected into a blastocyst ofan animal (e.g., a mouse) to form aggregation chimeras (see e.g.,Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term. Progeny harboringthe homologously recombined DNA in their germ cells can be used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA by germline transmission of the transgene. Methods forconstructing homologous recombination nucleic acid molecules, e.g.,vectors, or homologous recombinant animals are described further inBradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and in PCTInternational Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO93/04169 by Berns et al.

A transgenic animal of the invention can be created by introducing aPLTR-1-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection or retroviral infection, and allowingthe oocyte to develop in a pseudopregnant female foster animal. ThePLTR-1 cDNA sequence of SEQ ID NO:19 can be introduced as a transgeneinto the genome of a non-human animal. Alternatively, a non-humanhomologue of a human PLTR-1 gene, such as a rat or mouse PLTR-1 gene,can be used as a transgene. Alternatively, a PLTR-1 gene homologue, suchas another PLTR-1 family member, can be isolated based on hybridizationto the PLTR-1 cDNA sequences of SEQ ID NO:19 or 21, (described furtherin subsection I above) and used as a transgene. Intronic sequences andpolyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to aPLTR-1 transgene to direct expression of a PLTR-1 protein to particularcells. Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of a PLTR-1 transgene in its genome and/or expression of PLTR-1mRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene encoding a PLTR-1protein can further be bred to other transgenic animals carrying othertransgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a PLTR-1 gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the PLTR-1 gene. The PLTR-1 gene can be a humangene (e.g., the cDNA of SEQ ID NO:21), but more preferably, is anon-human homologue of a human PLTR-1 gene (e.g., a cDNA isolated bystringent hybridization with the nucleotide sequence of SEQ ID NO:19),For example, a mouse PLTR-1 gene can be used to construct a homologousrecombination nucleic acid molecule, e.g., a vector, suitable foraltering an endogenous PLTR-1 gene in the mouse genome. In a preferredembodiment, the homologous recombination nucleic acid molecule isdesigned such that, upon homologous recombination, the endogenous PLTR-1gene is functionally disrupted (i.e., no longer encodes a functionalprotein; also referred to as a “knock out” vector). Alternatively, thehomologous recombination nucleic acid molecule can be designed suchthat, upon homologous recombination, the endogenous PLTR-1 gene ismutated or otherwise altered but still encodes functional protein (e.g.,the upstream regulatory region can be altered to thereby alter theexpression of the endogenous PLTR-1 protein). In the homologousrecombination nucleic acid molecule, the altered portion of the PLTR-1gene is flanked at its 5′ and 3′ ends by additional nucleic acidsequence of the PLTR-1 gene to allow for homologous recombination tooccur between the exogenous PLTR-1 gene carried by the homologousrecombination nucleic acid molecule and an endogenous PLTR-1 gene in acell, e.g., an embryonic stem cell. The additional flanking PLTR-1nucleic acid sequence is of sufficient length for successful homologousrecombination with the endogenous gene. Typically, several kilobases offlanking DNA (both at the 5′ and 3′ ends) are included in the homologousrecombination nucleic acid molecule (see, e.g., Thomas, K. R. andCapecchi, M. R. (1987) Cell 51:503 for a description of homologousrecombination vectors). The homologous recombination nucleic acidmolecule is introduced into a cell, e.g., an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced PLTR-1 genehas homologously recombined with the endogenous PLTR-1 gene are selected(see e.g., Li, E. et al. (1992) Cell 69:915). The selected cells canthen be injected into a blastocyst of an animal (e.g., a mouse) to formaggregation chimeras (see e.g., Bradley, A., in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, Robertson, E. J. ed. (IRL,Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted intoa suitable pseudopregnant female foster animal and the embryo brought toterm. Progeny harboring the homologously recombined DNA in their germcells can be used to breed animals in which all cells of the animalcontain the homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination nucleicacid molecules, e.g., vectors, or homologous recombinant animals aredescribed further in Bradley, A. (1991) Curr. Opin. Biotechnol.2:823-829 and in PCT International Publication Nos.: WO 90/11354 by LeMouellec et al.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstraet al.; and WO 93/04169 by Berns et al.

A transgenic animal of the invention can be created by introducing aTFM-2 and/or TFM-3-encoding nucleic acid into the male pronuclei of afertilized oocyte, e.g., by microinjection, retroviral infection, andallowing the oocyte to develop in a pseudopregnant female foster animal.The TFM-2 and/or TFM-3 cDNA sequence of SEQ ID NO:27 or SEQ ID NO:30 canbe introduced as a transgene into the genome of a non-human animal.Alternatively, a nonhuman homologue of a human TFM-2 and/or TFM-3 gene,such as a mouse or rat TFM-2 and/or TFM-3 gene, can be used as atransgene. Alternatively, a TFM-2 and/or TFM-3 gene homologue, such asanother TFM-2 and/or TFM-3 family member, can be isolated based onhybridization to the TFM-2 and/or TFM-3 cDNA sequences of SEQ ID NO:27,29, 30, or 32 (described further in subsection I above) and used as atransgene. Intronic sequences and polyadenylation signals can also beincluded in the transgene to increase the efficiency of expression ofthe transgene. A tissue-specific regulatory sequence(s) can be operablylinked to a TFM-2 and/or TFM-3 transgene to direct expression of a TFM-2and/or TFM-3 polypeptide to particular cells. Methods for generatingtransgenic animals via embryo manipulation and microinjection,particularly animals such as mice, have become conventional in the artand are described, for example, in U.S. Pat. Nos. 4,736,866 and4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner etal. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods areused for production of other transgenic animals. A transgenic founderanimal can be identified based upon the presence of a TFM-2 and/or TFM-3transgene in its genome and/or expression of TFM-2 and/or TFM-3 mRNA intissues or cells of the animals. A transgenic founder animal can then beused to breed additional animals carrying the transgene. Moreover,transgenic animals carrying a transgene encoding a TFM-2 and/or TFM-3polypeptide can further be bred to other transgenic animals carryingother transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a TFM-2 and/or TFM-3 gene into which adeletion, addition or substitution has been introduced to thereby alter,e.g., functionally disrupt, the TFM-2 and/or TFM-3 gene. The TFM-2and/or TFM-3 gene can be a human gene (e.g., the cDNA of SEQ ID NO:29 or32), but more preferably, is a non-human homologue of a human TFM-2and/or TFM-3 gene (e.g., a cDNA isolated by stringent hybridization withthe nucleotide sequence of SEQ ID NO:27 or 30). For example, a mouseTFM-2 and/or TFM-3 gene can be used to construct a homologousrecombination nucleic acid molecule, e.g., a vector, suitable foraltering an endogenous TFM-2 and/or TFM-3 gene in the mouse genome. In apreferred embodiment, the homologous recombination nucleic acid moleculeis designed such that, upon homologous recombination, the endogenousTFM-2 and/or TFM-3 gene is functionally disrupted (i.e., no longerencodes a functional protein; also referred to as a “knock out” vector).Alternatively, the homologous recombination nucleic acid molecule can bedesigned such that, upon homologous recombination, the endogenous TFM-2and/or TFM-3 gene is mutated or otherwise altered but still encodesfunctional polypeptide (e.g., the upstream regulatory region can bealtered to thereby alter the expression of the endogenous TFM-2 and/orTFM-3 polypeptide). In the homologous recombination nucleic acidmolecule, the altered portion of the TFM-2 and/or TFM-3 gene is flankedat its 5′ and 3′ ends by additional nucleic acid sequence of the TFM-2and/or TFM-3 gene to allow for homologous recombination to occur betweenthe exogenous TFM-2 and/or TFM-3 gene carried by the homologousrecombination nucleic acid molecule and an endogenous TFM-2 and/or TFM-3gene in a cell, e.g., an embryonic stem cell. The additional flankingTFM-2 and/or TFM-3 nucleic acid sequence is of sufficient length forsuccessful homologous recombination with the endogenous gene. Typically,several kilobases of flanking DNA (both at the 5′ and 3′ ends) areincluded in the homologous recombination nucleic acid molecule (see,e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for adescription of homologous recombination vectors). The homologousrecombination nucleic acid molecule is introduced into a cell, e.g., anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced TFM-2 and/or TFM-3 gene has homologously recombined withthe endogenous TFM-2 and/or TFM-3 gene are selected (see e.g., Li, E. etal. (1992) Cell 69:915). The selected cells can then injected into ablastocyst of an animal (e.g., a mouse) to form aggregation chimeras(see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: APractical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp.113-152). A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term.Progeny harboring the homologously recombined DNA in their germ cellscan be used to breed animals in which all cells of the animal containthe homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination nucleicacid molecules, e.g., vectors, or homologous recombinant animals aredescribed further in Bradley, A. (1991) Current Opinion in Biotechnology2:823-829 and in PCT International Publication Nos.: WO 90/11354 by LeMouellec et al.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstraet al.; and WO 93/04169 by Berns et al.

A transgenic animal of the invention can be created by introducing a67118, 67067, and/or 62092-encoding nucleic acid into the male pronucleiof a fertilized oocyte, e.g., by microinjection, retroviral infection,and allowing the oocyte to develop in a pseudopregnant female fosteranimal. The 67118, 67067, and/or 62092 cDNA sequence of SEQ ID NO:33,36, or 39 can be introduced as a transgene into the genome of anon-human animal. Alternatively, a nonhuman homologue of a human 67118,67067, and/or 62092 gene, such as a mouse or rat 67118, 67067, and/or62092 gene, can be used as a transgene. Alternatively, a 67118, 67067,and/or 62092 gene homologue, such as another 67118, 67067, and/or 62092family member, can be isolated based on hybridization to the 67118,67067, and/or 62092 cDNA sequences of SEQ ID NO:33, 35, 36, 38, 39, or41, (described further in subsection I above) and used as a transgene.Intronic sequences and polyadenylation signals can also be included inthe transgene to increase the efficiency of expression of the transgene.A tissue-specific regulatory sequence(s) can be operably linked to a67118, 67067, and/or 62092 transgene to direct expression of a 67118,67067, and/or 62092 polypeptide to particular cells. Methods forgenerating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of a 67118, 67067, and/or 62092 transgene in its genome and/orexpression of 67118, 67067, and/or 62092 mRNA in tissues or cells of theanimals. A transgenic founder animal can then be used to breedadditional animals carrying the transgene. Moreover, transgenic animalscarrying a transgene encoding a 67118, 67067, and/or 62092 polypeptidecan further be bred to other transgenic animals carrying othertransgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a 67118, 67067, and/or 62092 gene intowhich a deletion, addition or substitution has been introduced tothereby alter, e.g., functionally disrupt, the 67118, 67067, and/or62092 gene. The 67118, 67067, and/or 62092 gene can be a human gene(e.g., the cDNA of SEQ ID NO:33, 36, or 39, respectively), but morepreferably, is a non-human homologue of a human 67118, 67067, and/or62092 gene (e.g., a cDNA isolated by stringent hybridization with thenucleotide sequence of SEQ II) NO:33, 36, or 39). For example, a mouse67118, 67067, and/or 62092 gene can be used to construct a homologousrecombination nucleic acid molecule, e.g., a vector, suitable foraltering an endogenous 67118, 67067, and/or 62092 gene in the mousegenome. In a preferred embodiment, the homologous recombination nucleicacid molecule is designed such that, upon homologous recombination, theendogenous 67118, 67067, and/or 62092 gene is functionally disrupted(i.e., no longer encodes a functional protein; also referred to as a“knock out” vector). Alternatively, the homologous recombination nucleicacid molecule can be designed such that, upon homologous recombination,the endogenous 67118, 67067, and/or 62092 gene is mutated or otherwisealtered but still encodes functional polypeptide (e.g., the upstreamregulatory region can be altered to thereby alter the expression of theendogenous 67118, 67067, and/or 62092 polypeptide). In the homologousrecombination nucleic acid molecule, the altered portion of the 67118,67067, and/or 62092 gene is flanked at its 5′ and 3′ ends by additionalnucleic acid sequence of the 67118, 67067, and/or 62092 gene to allowfor homologous recombination to occur between the exogenous 67118,67067, and/or 62092 gene carried by the homologous recombination nucleicacid molecule and an endogenous 67118, 67067, and/or 62092 gene in acell, e.g., an embryonic stem cell. The additional flanking 67118,67067, and/or 62092 nucleic acid sequence is of sufficient length forsuccessful homologous recombination with the endogenous gene. Typically,several kilobases of flanking DNA (both at the 5′ and 3′ ends) areincluded in the homologous recombination nucleic acid molecule (see,e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for adescription of homologous recombination vectors). The homologousrecombination nucleic acid molecule is introduced into a cell, e.g., anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced 67118, 67067, and/or 62092 gene has homologouslyrecombined with the endogenous 67118, 67067, and/or 62092 gene areselected (see e.g., Li, E. et al. (1992) Cell 69:915). The selectedcells can then injected into a blastocyst of an animal (e.g., a mouse)to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomasand Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed.(IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then beimplanted into a suitable pseudopregnant female foster animal and theembryo brought to term. Progeny harboring the homologously recombinedDNA in their germ cells can be used to breed animals in which all cellsof the animal contain the homologously recombined DNA by germlinetransmission of the transgene. Methods for constructing homologousrecombination nucleic acid molecules,. e.g., vectors, or homologousrecombinant animals are described further in Bradley, A. (1991) CurrentOpinion in Biotechnology 2:823-829 and in PCT International PublicationNos.: WO 90/11354 by Le Mouellec et al.; WO 91/01140 by Smithies et al.;WO 92/0968 by Zijlstra et al.; and WO 93/04169 by Berns et al.

A transgenic animal of the invention can be created by introducing aHAAT-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection or retroviral infection, and allowingthe oocyte to develop in a pseudopregnant female foster animal. The HAATcDNA sequence of SEQ ID NO:51 can be introduced as a transgene into thegenome of a non-human animal. Alternatively, a non-human homologue of ahuman HAAT gene, such as a rat or mouse HAAT gene, can be used as atransgene. Alternatively, a HAAT gene homologue, such as another HAATfamily member, can be isolated based on hybridization to the HAAT cDNAsequences of SEQ ID NO:51 or 53, (described further in subsection Iabove) and used as a transgene. Intronic sequences and polyadenylationsignals can also be included in the transgene to increase the efficiencyof expression of the transgene. A tissue-specific regulatory sequence(s)can be operably linked to a HAAT transgene to direct expression of aHAAT protein to particular cells. Methods for generating transgenicanimals via embryo manipulation and microinjection, particularly animalssuch as mice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder etal., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986). Similar methods are used for productionof other transgenic animals. A transgenic founder animal can beidentified based upon the presence of a HAAT transgene in its genomeand/or expression of HAAT mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding a HAAT protein can further be bred to othertransgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a HAAT gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the HAAT gene. The HAAT gene can be a human gene(e.g., the cDNA of SEQ ID NO:53), but more preferably, is a non-humanhomologue of a human HAAT gene (e.g., a cDNA isolated by stringenthybridization with the nucleotide sequence of SEQ ID NO:51), Forexample, a mouse HAAT gene can be used to construct a homologousrecombination nucleic acid molecule, e.g., a vector, suitable foraltering an endogenous HAAT gene in the mouse genome. In a preferredembodiment, the homologous recombination nucleic acid molecule isdesigned such that, upon homologous recombination, the endogenous HAATgene is functionally disrupted (i.e., no longer encodes a functionalprotein; also referred to as a “knock out” vector). Alternatively, thehomologous recombination nucleic acid molecule can be designed suchthat, upon homologous recombination, the endogenous HAAT gene is mutatedor otherwise altered but still encodes functional protein (e.g., theupstream regulatory region can be altered to thereby alter theexpression of the endogenous HAAT protein). In the homologousrecombination nucleic acid molecule, the altered portion of the HAATgene is flanked at its 5′ and 3′ ends by additional nucleic acidsequence of the HAAT gene to allow for homologous recombination to occurbetween the exogenous HAAT gene carried by the homologous recombinationnucleic acid molecule and an endogenous HAAT gene in a cell, e.g., anembryonic stem cell. The additional flanking HAAT nucleic acid sequenceis of sufficient length for successful homologous recombination with theendogenous gene. Typically, several kilobases of flanking DNA (both atthe 5′ and 3′ ends) are included in the homologous recombination nucleicacid molecule (see, e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell51:503 for a description of homologous recombination vectors). Thehomologous recombination nucleic acid molecule is introduced into acell, e.g., an embryonic stem cell line (e.g., by electroporation) andcells in which the introduced HAAT gene has homologously recombined withthe endogenous HAAT gene are selected (see e.g., Li, E. et al. (1992)Cell 69:915). The selected cells can then be injected into a blastocystof an animal (e.g., a mouse) to form aggregation chimeras (see e.g.,Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, Robertson, E. J. ed. (IRL, Oxford, 1987) pp. 113-152). Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term. Progeny harboringthe homologously recombined DNA in their germ cells can be used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA by germline transmission of the transgene. Methods forconstructing homologous recombination nucleic acid molecules, e.g.,vectors, or homologous recombinant animals are described further inBradley, A. (1991) Curr. Opin. Biotechnol. 2:823-829 and in PCTInternational Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO93/04169 by Berns et al.

A transgenic animal of the invention can be created by introducing anHST-4- and/or an HST-5-encoding nucleic acid into the male pronuclei ofa fertilized oocyte, e.g., by microinjection, retroviral infection, andallowing the oocyte to develop in a pseudopregnant female foster animal.The HST-4 and/or HST-5 cDNA sequence of SEQ ID NO:54 or 57 can beintroduced as a transgene into the genome of a non-human animal.Alternatively, a nonhuman homologue of a human HST-4 and/or HST-5 gene,such as a mouse or rat HST-4 and/or HST-5 gene, can be used as atransgene. Alternatively, an HST-4 and/or an HST-5 gene homologue, suchas another HST-4 and/or HST-5 family member, can be isolated based onhybridization to the HST-4 and/or HST-5 cDNA sequences of SEQ ID NO:54,56, 57, or 59, (described further in subsection I above) and used as atransgene. Intronic sequences and polyadenylation signals can also beincluded in the transgene to increase the efficiency of expression ofthe transgene. A tissue-specific regulatory sequence(s) can be operablylinked to an HST-4 and/or an HST-5 transgene to direct expression of anHST-4 and/or an HST-5 polypeptide to particular cells. Methods forgenerating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of an HST-4 and/or an HST-5 transgene in its genome and/orexpression of HST-4 and/or HST-5 mRNA in tissues or cells of theanimals. A transgenic founder animal can then be used to breedadditional animals carrying the transgene. Moreover, transgenic animalscarrying a transgene encoding an HST-4 and/or an HST-5 polypeptide canfurther be bred to other transgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of an HST-4 and/or an HST-5 gene into whicha deletion, addition or substitution has been introduced to therebyalter, e.g., functionally disrupt, the HST-4 and/or the HST-5 gene. TheHST-4 and/or the HST-5 gene can be a human gene (e.g., the cDNA of SEQID NO:56 or 59), but more preferably, is a non-human homologue of ahuman HST-4 and/or HST-5 gene (e.g., a cDNA isolated by stringenthybridization with the nucleotide sequence of SEQ ID NO:54 or 57). Forexample, a mouse HST-4 and/or HST-5 gene can be used to construct ahomologous recombination nucleic acid molecule, e.g., a vector, suitablefor altering an endogenous HST-4 and/or HST-5 gene in the mouse genome.In a preferred embodiment, the homologous recombination nucleic acidmolecule is designed such that, upon homologous recombination, theendogenous HST-4 and/or HST-5 gene is functionally disrupted (i.e., nolonger encodes a functional protein; also referred to as a “knock out”vector). Alternatively, the homologous recombination nucleic acidmolecule can be designed such that, upon homologous recombination, theendogenous HST-4 and/or HST-5 gene is mutated or otherwise altered butstill encodes functional polypeptide (e.g., the upstream regulatoryregion can be altered to thereby alter the expression of the endogenousHST-4 and/or HST-5 polypeptide). In the homologous recombination nucleicacid molecule, the altered portion of the HST-4 and/or the HST-5 gene isflanked at its 5′ and 3′ ends by additional nucleic acid sequence of theHST-4 and/or the HST-5 gene to allow for homologous recombination tooccur between the exogenous HST-4 and/or HST-5 gene carried by thehomologous recombination nucleic acid molecule and an endogenous HST-4and/or HST-5 gene in a cell, e.g., an embryonic stem cell. Theadditional flanking HST-4 and/or HST-5 nucleic acid sequence is ofsufficient length for successful homologous recombination with theendogenous gene. Typically, several kilobases of flanking DNA (both atthe 5′ and 3′ ends) are included in the homologous recombination nucleicacid molecule (see, e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell51:503 for a description of homologous recombination vectors). Thehomologous recombination nucleic acid molecule is introduced into acell, e.g., an embryonic stem cell line (e.g., by electroporation) andcells in which the introduced HST-4 and/or HST-5 gene has homologouslyrecombined with the endogenous HST-4 and/or HST-5 gene are selected (seee.g., Li, E. et al. (1992) Cell 69:915). The selected cells can theninjected into a blastocyst of an animal (e.g., a mouse) to formaggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted intoa suitable pseudopregnant female foster animal and the embryo brought toterm. Progeny harboring the homologously recombined DNA in their germcells can be used to breed animals in which all cells of the animalcontain the homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination nucleicacid molecules, e.g., vectors, or homologous recombinant animals aredescribed further in Bradley, A. (1991) Current Opinion in Biotechnology2:823-829 and in PCT International Publication Nos.: WO 90/11354 by LeMouellec et al.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstraet al.; and WO 93/04169 by Berns et al.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355. If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. (1997)Nature 385:810-813 and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyte and then transferred to pseudopregnant femalefoster animal. The offspring borne of this female foster animal will bea clone of the animal from which the cell, e.g., the somatic cell, isisolated.

IV. Pharmaceutical Compositions

The MTP-1 nucleic acid molecules, fragments of MTP-1 proteins, andanti-MTP-1 antibodies (also referred to herein as “active compounds”) ofthe invention can be incorporated into pharmaceutical compositionssuitable for administration. Such compositions typically comprise thenucleic acid molecule, protein, or antibody and a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The OAT, PLTR-1 and/or HAAT nucleic acid molecules, OAT, PLTR-1 and/orHAAT proteins, fragments thereof, anti-OAT, anti-PLTR-1 and/or anti-HAATantibodies, and OAT, PLTR-1 and/or HAAT modulators (also referred toherein as “active compounds”) of the invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise the nucleic acid molecule, protein, orantibody and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” is intended to includeany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

The HST-1, TP-2, TFM-2 and/or TFM-3 nucleic acid molecules, fragments ofHST-1, TP-2, TFM-2 and/or TFM-3 polypeptides, and anti-HST-1, anti-TP-2,anti-TFM-2 and/or anti-TFM-3 antibodies (also referred to herein as“active compounds”) of the invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise the nucleic acid molecule, polypeptide,or antibody and a pharmaceutically acceptable carrier. As used hereinthe language “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

The 67118, 67067, 62092, HST-4 and/or the HST-5 nucleic acid molecules,fragments of 67118, 67067, 62092, HST-4 and/or HST-5 polypeptides,anti-67118, anti-67067, anti-62092, anti-HST-4 and/or anti-HST-5antibodies, and/or 67118, 67067, 62092, HST-4 modulators and/or HST-5modulators (also referred to herein as “active compounds”) of theinvention can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the nucleicacid molecule, polypeptide, or antibody and a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a fragment of an MTP-1, an OAT, an HST-1, a TP-2, aPLTR-1, a TFM-2, a TFM-3, a 67118, a 67067, a 62092, a HAAT, an HST-4and/or an HST-5 polypeptide or an anti-MTP-1, anti-OAT, anti-HST-1,anti-TP-2, anti-PLTR-1, anti-TFM-2, anti-TFM-3, anti-67118, anti-67067,anti-62092, anti- HAAT, anti-HST-4 and/or anti-HST-5 antibody) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of polypeptide(i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg bodyweight, preferably about 0.01 to 25 mg/kg body weight, more preferablyabout 0.1 to 20 mg/kg body weight, and even more preferably about 1 to10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg bodyweight. The skilled artisan will appreciate that certain factors mayinfluence the dosage required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a polypeptide or antibody caninclude a single treatment or, preferably, can include a series oftreatments.

In a preferred example, a subject is treated with antibody orpolypeptide in the range of between about 0.1 to 20 mg/kg body weight,one time per week for between about 1 to 10 weeks, preferably between 2to 8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. It will also be appreciated thatthe effective dosage of antibody or polypeptide used for treatment mayincrease or decrease over the course of a particular treatment. Changesin dosage may result and become apparent from the results of diagnosticassays as described herein.

The present invention encompasses agents which modulate expression oractivity. An agent may, for example, be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e.,. including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds. It is understood that appropriate doses of smallmolecule agents depends upon a number of factors within the ken of theordinarily skilled physician, veterinarian, or researcher. The dose(s)of the small molecule will vary, for example, depending upon theidentity, size, and condition of the subject or sample being treated,further depending upon the route by which the composition is to beadministered, if applicable, and the effect which the practitionerdesires the small molecule to have upon the nucleic acid or polypeptideof the invention.

Exemplary doses include milligram or microgram amounts of the smallmolecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram. It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. Such appropriate doses may be determined usingthe assays described herein. When one or more of these small moleculesis to be administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

In certain embodiments of the invention, a modulator of OAT, PLTR-1 orHAAT activity is administered in combination with other agents (e.g., asmall molecule), or in conjunction with another, complementary treatmentregime. For example, in one embodiment, a modulator of OAT, PLTR-1 orHAAT activity is used to treat OAT, PLTR-1 or HAAT associated disorder.Accordingly, modulation of OAT, PLTR-1 or HAAT activity may be used inconjunction with, for example, another agent used to treat the disorder.

Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologues thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, alpha-interferon, beta-interferon, nerve growthfactor, platelet derived growth factor, tissue plasminogen activator;or, biological response modifiers such as, for example, lymphokines,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocytecolony stimulating factor (“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate asdescribed by Segal in U.S. Pat. No. 4,676,980.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

V. Uses and Methods of the Invention

A. MTP-1

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) predictive medicine (e.g., diagnostic assays,prognostic assays, monitoring clinical trials, and pharmacogenetics);and c) methods of treatment (e.g., therapeutic and prophylactic). Asdescribed herein, an MTP-1 protein of the invention has one or more ofthe following activities: 1) modulates the import and export ofmolecules from cells, e.g., lipids, hormones, ions, cytokines,neurotransmitters, and metabolites, 2) modulates intra- or intercellularsignaling, 3) modulates removal of potentially harmful compounds fromthe cell, or facilitate the compartmentalization of these molecules intoa sequestered intracellular space (e.g., the peroxisome), and 4)modulates transport of biological molecules across membranes, e.g., theplasma membrane, or the membrane of the mitochondrion, the peroxisome,the lysosome, the endoplasmic reticulum, the nucleus, or the vacuole.

The isolated nucleic acid molecules of the invention can be used, forexample, to express MTP-1 protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect MTP-1mRNA (e.g., in a biological sample) or a genetic alteration in an MTP-1gene, and to modulate MTP-1 activity, as described further below. TheMTP-1 proteins can be used to treat disorders characterized byinsufficient or excessive production of an MTP-1 substrate or productionof MTP-1 inhibitors. In addition, the MTP-1 proteins can be used toscreen for naturally occurring MTP-1 substrates, to screen for drugs orcompounds which modulate MTP-1 activity, as well as to treat disorderscharacterized by insufficient or excessive production of MTP-1 proteinor production of MTP-1 protein forms which have decreased, aberrant orunwanted activity compared to MTP-1 wild type protein, preferably atransporter-associated disorder. As used herein, a“transporter-associated disorder” includes a disorder, disease orcondition which is caused or characterized by a misregulation (e.g.,downregulation or upregulation) of a transporter-mediated activity.Transporter-associated disorders can detrimentally affect cellularfunctions such as inflammation, lipid metabolism, hematopoiesis,cellular proliferation, growth, differentiation, or migration, cellularregulation of homeostasis, inter- or intra-cellular communication;tissue function, such as cardiac function or musculoskeletal function;systemic responses in an organism, such as nervous system responses,hormonal responses (e.g., insulin response), or immune responses; andprotection of cells from toxic compounds (e.g., carcinogens, toxins,mutagens, and toxic byproducts of metabolic activity (e.g., reactiveoxygen species)).

Since MTP-1 is preferentially expressed in hematopoietic tissue such asbone marrow cells, MTP-1 molecules may be causatively linked tohematopoietic disorders, examples of which include disorders relating tothe proliferation, differentiation, and/or function of cells that appearin the bone marrow, e.g., stem cells (e.g., hematopoietic stem cells),and blood cells, e.g., erythrocytes, platelets, and leukocytes. Thus [x]nucleic acids, proteins, and modulators thereof can be used to treatbone marrow, blood, and hematopoietic associated diseases and disorders,e.g., acute myeloid leukemia, hemophilia, leukemia, anemia (e.g., sicklecell anemia), and thalassemia.

In another example, MTP-1 polypeptides, nucleic acids, and modulatorsthereof can be used to treat leukocytic disorders, such as leukopenias(e.g., neutropenia, monocytopenia, lymphopenia, and granulocytopenia),leukocytosis (e.g., granulocytosis, lymphocytosis, eosinophilia,monocytosis, acute and chronic lymphadenitis), malignant lymphomas(e.g., Non-Hodgkin's lymphomas, Hodgkin's lymphomas, leukemias,agnogenic myeloid metaplasia, multiple myeloma, plasmacytoma,Waldenstrom's macroglobulinemia, heavy-chain disease, monoclonalgammopathy, histiocytoses, eosinophilic granuloma, andangioimmunoblastic lymphadenopathy).

Since MTP-1 is homologous to known ABC transporter molecules, which areknown to be causatively linked to disorders related to lipid metabolism,MTP-1 molecules may be causatively linked to disorders related to lipidmetabolism, adipocyte function and adipocyte-related processes such as,e.g., obesity, regulation of body temperature, lipid metabolism,carbohydrate metabolism, body weight regulation, obesity, anorexianervosa, diabetes mellitus, unusual susceptibility or insensitivity toheat or cold, arteriosclerosis, atherosclerosis, atherogenesis anddisorders involving abnormal vascularization, e.g., vascularization ofsolid tumors.

Examples of transporter-associated disorders also include immunologicaldisorders such as autoimmune disorders (e.g., arthritis, graft rejection(e.g., allograft rejection), T cell disorders (e.g., AIDS)), immunedeficiency disorders, e.g., congenital X-linked infantilehypogammaglobulinemia, transient hypogammaglobulinemia, common variableimmunodeficiency, selective IgA deficiency, chronic mucocutaneouscandidiasis, or severe combined immunodeficiency. Transporter-relateddisorders also include inflammatory disorders pertaining to,characterized by, causing, resulting from, or becoming affected byinflammation. Examples of inflammatory diseases or disorders include,without limitation, asthma, lung inflammation, chronic granulomatousdiseases such as tuberculosis, leprosy, sarcoidosis, silicosis andschistosomiasis, nephritis, amyloidosis, rheumatoid arthritis,ankylosing sponduylitis, chronic bronchitis, scleroderma, lupus,polymyositis, appendicitis, inflammatory bowel disease, ulcers,Sjorgen's syndrome, Reiter's syndrome, psoriasis, pelvic inflammatorydisease, orbital inflammatory disease, thrombotic disease, andinappropriate allergic responses to environmental stimuli such as poisonivy, pollen, insect stings and certain foods, including atopicdermatitis and contact dermatitis.

Examples of transporter-associated disorders also include CNS disorderssuch as cognitive and neurodegenerative disorders, examples of whichinclude, but are not limited to, Alzheimer's disease, dementias relatedto Alzheimer's disease (such as Pick's disease), Parkinson's and otherLewy diffuse body diseases, senile dementia, Huntington's disease,Gilles de la Tourette's syndrome, multiple sclerosis, amyotrophiclateral sclerosis, progressive supranuclear palsy, epilepsy, andJakob-Creutzfieldt disease; autonomic function disorders such ashypertension and sleep disorders, and neuropsychiatric disorders, suchas depression, schizophrenia, schizoaffective disorder, korsakoff'spsychosis, mania, anxiety disorders, or phobic disorders; learning ormemory disorders, e.g., amnesia or age-related memory loss, attentiondeficit disorder, dysthymic disorder, major depressive disorder, mania,obsessive-compulsive disorder, psychoactive substance use disorders,anxiety, phobias, panic disorder, as well as bipolar affective disorder,e.g., severe bipolar affective (mood) disorder (BP-1), and bipolaraffective neurological disorders, e.g., migraine and obesity. FurtherCNS-related disorders include, for example, those listed in the AmericanPsychiatric Association's Diagnostic and Statistical manual of MentalDisorders (DSM), the most current version of which is incorporatedherein by reference in its entirety.

Further examples of transporter-associated disorders includecardiac-related disorders. Cardiovascular system disorders in which theMTP-1 molecules of the invention may be directly or indirectly involvedinclude arteriosclerosis, ischemia reperfusion injury, restenosis,arterial inflammation, vascular wall remodeling, ventricular remodeling,rapid ventricular pacing, coronary microembolism, tachycardia,bradycardia, pressure overload, aortic bending, coronary arteryligation, vascular heart disease, atrial fibrilation, Jervell syndrome,Lange-Nielsen syndrome, long-QT syndrome, congestive heart failure,sinus node dysfunction, angina, heart failure, hypertension, atrialfibrillation, atrial flutter, dilated cardiomyopathy, idiopathiccardiomyopathy, myocardial infarction, coronary artery disease, coronaryartery spasm, and arrhythmia. MTP-1-mediated or related disorders alsoinclude disorders of the musculoskeletal system such as paralysis andmuscle weakness, e.g., ataxia, myotonia, and myokymia.

Transporter disorders also include cellular proliferation, growth,differentiation, or migration disorders. Cellular proliferation, growth,differentiation, or migration disorders include those disorders thataffect cell proliferation, growth, differentiation, or migrationprocesses. As used herein, a “cellular proliferation, growth,differentiation, or migration process” is a process by which a cellincreases in number, size or content, by which a cell develops aspecialized set of characteristics which differ from that of othercells, or by which a cell moves closer to or further from a particularlocation or stimulus. The MTP-1 molecules of the present invention areinvolved in signal transduction mechanisms, which are known to beinvolved in cellular growth, differentiation, and migration processes.Thus, the MTP-1 molecules may modulate cellular growth, differentiation,or migration, and may play a role in disorders characterized byaberrantly regulated growth, differentiation, or migration. Suchdisorders include cancer, e.g., carcinoma, sarcoma, or leukemia; tumorangiogenesis and metastasis; skeletal dysplasia; hepatic disorders; andhematopoietic and/or myeloproliferative disorders.

MTP-1-associated or related disorders also include hormonal disorders,such as conditions or diseases in which the production and/or regulationof hormones in an organism is aberrant. Examples of such disorders anddiseases include type I and type II diabetes mellitus, pituitarydisorders (e.g., growth disorders), thyroid disorders (e.g.,hypothyroidism or hyperthyroidism), and reproductive or fertilitydisorders (e.g., disorders which affect the organs of the reproductivesystem, e.g., the prostate gland, the uterus, or the vagina; disorderswhich involve an imbalance in the levels of a reproductive hormone in asubject; disorders affecting the ability of a subject to reproduce; anddisorders affecting secondary sex characteristic development, e.g.,adrenal hyperplasia).

MTP-1-associated or related disorders also include disorders affectingtissues in which MTP-1 protein is expressed.

1. MTP-1 Screening Assays:

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules (organic orinorganic) or other drugs) which bind to MTP-1 proteins, have astimulatory or inhibitory effect on, for example, MTP-1 expression orMTP-1 activity, or have a stimulatory or inhibitory effect on, forexample, the transport (e.g., import or export) of an MTP-1 substrate(e.g., cytotoxic substances, ions, peptides, metabolites).

These assays are designed to identify compounds that bind to a MTP-1protein, bind to other inter- or extra-cellular proteins that interactwith a MTP-1 protein, and/or interfere with the interaction of the MTP-1protein with other inter- or extra-cellular proteins. For example, inthe case of the MTP-1 protein, which is protein that is capable ofmembrane transport, such techniques can be used to identify ligands forsuch a protein. A MTP-1 protein modulator can, for example, be used toameliorate diseases or disorders related to transmembrane lipidtransport and/or hematopoietic cells. Such compounds may include, butare not limited to MTP-1 peptides, anti-MTP-1 antibodies, or smallorganic or inorganic compounds. Such compounds may also include othercellular proteins or peptides.

Compounds identified via assays such as those described herein may beuseful, for example, for ameliorating hematopoietic and/or immunologicaland/or lipid metabolism-related diseases or disorders. In instanceswhereby a hematopoietic and/or immunological and/or lipidmetabolism-related disease condition results from an overall lower levelof MTP-1 gene expression and/or MTP-1 protein in a cell or tissue,compounds that interact with the MTP-1 protein may include compoundswhich accentuate or amplify the activity of the bound MTP-1 protein.Such compounds would bring about an effective increase in the level ofMTP-1 protein activity, thus ameliorating symptoms.

In other instances, mutations within the MTP-1 gene may cause aberranttypes or excessive amounts of MTP-1 proteins to be made which have adeleterious effect that leads to a hematopoietic and/or immunologicaland/or lipid metabolism-related disease or disorder. Similarly,physiological conditions may cause an excessive increase in MTP-1 geneexpression leading to a hematopoietic and/or immunological and/or lipidmetabolism-related disease or disorder. In such cases, compounds thatbind to a MTP-1 protein may be identified that inhibit the activity ofthe MTP-1 protein. Assays for testing the effectiveness of compoundsidentified by techniques such as those described in this section arediscussed herein.

In one embodiment, the invention provides assays for screening candidateor test compounds which are capable of binding to and/or beingtransported by an MTP-1 protein or polypeptide or biologically activeportion thereof. In another embodiment, the invention provides assaysfor screening candidate or test compounds which bind to or modulate theactivity of an MTP-1 protein or polypeptide or biologically activeportion thereof, e.g., which modulate the ability of an MTP-1 protein totransport an MTP-1 substrate (e.g., a cytotoxic substance, an ion, apeptide, a metabolite). The test compounds of the present invention canbe obtained using any of the numerous approaches in combinatoriallibrary methods known in the art, including: biological libraries;spatially addressable parallel solid phase or solution phase libraries;synthetic library methods requiring deconvolution; the ‘one-beadone-compound’ library method; and synthetic library methods usingaffinity chromatography selection. The biological library approach islimited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37: 1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner USP '409), plasmids (Cull etal. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scottand Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses an MTP-1 protein or biologically active portion thereof iscontacted with a test compound and the ability of the test compound tomodulate MTP-1 activity is determined. Determining the ability of thetest compound to modulate MTP-1 activity can be accomplished bymonitoring, for example, the transport of an MTP-1 substrate into or outof a cell which expresses MTP-1. The cell, for example, can be ofmammalian origin, e.g., a murine or human cell. The ability of the testcompound to modulate MTP-1 transport of a substrate (e.g., cytotoxicsubstances, ions, peptides, metabolites) or to bind to MTP-1 can also bedetermined. Determining the ability of the test compound to modulateMTP-1 transport of a substrate (e.g., cytotoxic substances, ions,peptides, metabolites) can be accomplished, for example, by coupling theMTP-1 substrate with a radioisotope or enzymatic label such thattransport of the MTP-1 substrate by MTP-1 can be determined by detectingthe labeled MTP-1 substrate (e.g., in the cell, extracellularly, orintercompartmentally). Determining the ability of the test compound tobind MTP-1 can be accomplished, for example, by coupling the compoundwith a radioisotope or enzymatic label such that binding of the compoundto MTP-1 can be determined by detecting the labeled MTP-1 compound, forexample, complexed to MTP-1 in a cell membrane. For example, compounds(e.g., MTP-1 substrates) can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H,either directly or indirectly, and the radioisotope detected by directcounting of radioemission or by scintillation counting. Alternatively,compounds can be enzymatically labeled with, for example, horseradishperoxidase, alkaline phosphatase, or luciferase, and the enzymatic labeldetected by determination of conversion of an appropriate substrate toproduct.

It is also within the scope of this invention to determine the abilityof a compound (e.g., an MTP-1 substrate, e.g., cytotoxic substances,ions, peptides, metabolites) to interact with or to be transported byMTP-1 without the labeling of any of the interactants. For example, amicrophysiometer can be used to detect the interaction of a compoundwith MTP-1 without the labeling of either the compound or the MTP-1.McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a“microphysiometer” (e.g., Cytosensor) is an analytical instrument thatmeasures the rate at which a cell acidifies its environment using alight-addressable potentiometric sensor (LAPS). Changes in thisacidification rate can be used as an indicator of the interactionbetween a compound and MTP-1.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell which expresses or produces MTP-1 with an MTP-1substrate (e.g., a cytotoxic substance, an ion, a peptide, a metabolite)and a test compound and determining the ability of the test compound tomodulate (e.g., stimulate or inhibit) the activity (e.g., transport) orcellular location of the MTP-1 substrate molecule.

Determining the ability of the MTP-1 protein, or a biologically activefragment thereof, to bind to, interact with, or transport an MTP-1substrate (e.g., cytotoxic substances, ions, peptides, metabolites) canbe accomplished by one of the methods described above for determiningdirect binding. In a preferred embodiment, determining the ability ofthe MTP-1 protein to bind to, interact with, or transport an MTP-1substrate (e.g., cytotoxic substances, ions, peptides, metabolites) canbe accomplished by determining the activity or localization of thesubstrate molecule. For example, the activity of the substrate can bedetermined by detecting induction of a cellular response (i.e., changesin intracellular K⁺ levels), detecting a secondary or indirect activityof the substrate on a downstream molecule, detecting the induction of areporter gene (comprising a substrate-responsive regulatory elementoperatively linked to a nucleic acid encoding a detectable marker, e.g.,luciferase), detecting a substrate-regulated cellular response, ordetermining the localization of the substrate molecule. In otherembodiments, the assays described above are carried out in a cell-freecontext (e.g., in an artificial membrane, vesicle, or micellepreparation).

In one embodiment, an assay of the present invention is a cell-freeassay in which an MTP-1 protein or biologically active portion thereof(e.g., a portion which possesses the ability to transport or interactwith an MTP-1 substrate, e.g., a cytotoxic substance, an ion, a peptide,or a metabolite) is contacted with a test compound and the ability ofthe test compound to bind to the MTP-1 protein or biologically activeportion thereof is determined. Preferred biologically active portions ofthe MTP-1 proteins to be used in assays of the present invention includefragments which participate in interactions with non-MTP-1 molecules,e.g., fragments with high surface probability scores. Binding of thetest compound to the MTP-1 protein can be determined either directly orindirectly as described above. In a preferred embodiment, the assayincludes contacting the MTP-1 protein or biologically active portion(e.g., a portion which possesses the ability to transport or interactwith an MTP-1 substrate, e.g., a cytotoxic substance, an ion, a peptide,or a metabolite) thereof with a known compound which binds MTP-1 to forman assay mixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to interact with an MTP-1protein, wherein determining the ability of the test compound tointeract with an MTP-1 protein comprises determining the ability of thetest compound to preferentially bind to MTP-1 or biologically activeportion thereof as compared to the known compound.

In another embodiment, the assay is a cell-free assay in which an MTP-1protein or biologically active portion thereof (e.g., a portion whichpossesses the ability to transport or interact with an MTP-1 substrate,e.g., a cytotoxic substance, an ion, a peptide, or a metabolite) iscontacted with a test compound and the ability of the test compound tomodulate (e.g., stimulate or inhibit) the activity of the MTP-1 proteinor biologically active portion thereof is determined. Determining theability of the test compound to modulate the activity of an MTP-1protein can be accomplished, for example, by determining the ability ofthe MTP-1 protein to transport an MTP-1 substrate as described herein.Determining the ability of the MTP-1 protein to bind to an MTP-1substrate (e.g., cytotoxic substances, ions, peptides, metabolites) canalso be accomplished using a technology such as real-time BiomolecularInteraction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991)Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct.Biol. 5:699-705. As used herein, “BIA” is a technology for studyingbiospecific interactions in real time, without labeling any of theinteractants (e.g., BIAcore). Changes in the optical phenomenon ofsurface plasmon resonance (SPR) can be used as an indication ofreal-time reactions between biological molecules.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize MTP-1 (e.g., MTP-1 in acell, vesicle, or membrane preparation) MTP-1 protein can be immobilizedfor example on the surface of any vessel suitable for containingreactants. Examples of such vessels include microtitre plates, testtubes, and micro-centrifuge tubes. For example, an MTP-1 protein can beimmobilized utilizing conjugation of biotin and streptavidin.Biotinylated MTP-1 protein can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques known in the art (e.g.,biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized inthe wells of streptavidin-coated 96 well plates (Pierce Chemical).Alternatively, antibodies reactive with MTP-1 protein or targetmolecules but which do not interfere with activity of the MTP-1 proteincan be derivatized to the wells of the plate, and unbound MTP-1 proteintrapped in the wells by antibody conjugation.

In another embodiment, modulators of MTP-1 expression are identified ina method wherein a cell is contacted with a candidate compound and theexpression of MTP-1 mRNA or protein in the cell is determined. The levelof expression of MTP-1 mRNA or protein in the presence of the candidatecompound is compared to the level of expression of MTP-1 mRNA or proteinin the absence of the candidate compound. The candidate compound canthen be identified as a modulator of MTP-1 expression based on thiscomparison. For example, when expression of MTP-1 mRNA or protein isgreater (statistically significantly greater) in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator of MTP-1 mRNA or protein expression.Alternatively, when expression of MTP-1 mRNA or protein is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of MTP-1 mRNA or protein expression. The level of MTP-1 mRNAor protein expression in the cells can be determined by methodsdescribed herein for detecting MTP-1 mRNA or protein.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based assay or a cell free assay (e.g., anartificial membrane, micelle, or vesicle preparation), and the abilityof the agent to modulate the activity of an MTP-1 protein can beconfirmed in vivo, e.g., in an animal such as an animal model forcellular transformation and/or tumorigenesis.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., an MTP-1 modulating agent, an antisense MTP-1nucleic acid molecule, an MTP-1-specific antibody, or an MTP-1-bindingpartner) can be used in an animal model to determine the efficacy,toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal model to determine the mechanism of action of such an agent.Furthermore, this invention pertains to uses of novel agents identifiedby the above-described screening assays for treatments as describedherein. In one embodiment, the invention features a method of treating asubject having a hematopoietic and/or immunological and/or lipidmetabolism-related disease or disorder that involves administering tothe subject a MTP-1 modulator such that treatment occurs. In anotherembodiment, the invention features a method of treating a subject havinga hematopoietic and/or immunological and/or lipid metabolism-relateddisease, e.g., atherogenesis, that involves treating a subject with aMTP-1 modulator, such that treatment occurs. Preferred MTP-1 modulatorsinclude, but are not limited to, MTP-1 proteins or biologically activefragments, MTP-1 nucleic acid molecules, MTP-1 antibodies, ribozymes,and MTP-1 antisense oligonucleotides designed based on the MTP-1nucleotide sequences disclosed herein, as well as peptides, organic andnon-organic small molecules identified as being capable of modulatingMTP-1 expression and/or activity, for example, according to at least oneof the screening assays described herein.

Any of the compounds, including but not limited to compounds such asthose identified in the foregoing assay systems, may be tested for theability to ameliorate immunological disease or disorder symptoms.Cell-based and animal model-based assays for the identification ofcompounds exhibiting such an ability to ameliorate hematopoietic and/orimmunological and/or lipid metabolism-related disease or disordersystems are described herein.

In one aspect, cell-based systems, as described herein, may be used toidentify compounds which may act to ameliorate hematopoietic and/orimmunological and/or lipid metabolism-related disease or disordersymptoms. For example, such cell systems may be exposed to a compound,suspected of exhibiting an ability to ameliorate hematopoietic and/orimmunological and/or lipid metabolism-related disease or disordersymptoms, at a sufficient concentration and for a time sufficient toelicit such an amelioration of hematopoietic and/or immunological and/orlipid metabolism-related disease or disorder symptoms in the exposedcells. After exposure, the cells are examined to determine whether oneor more of the hematopoietic and/or immunological and/or lipidmetabolism-related disease or disorder cellular phenotypes has beenaltered to resemble a more normal or more wild type, non-hematopoieticand/or immunological and/or lipid metabolism-related disease or disorderphenotype. Cellular phenotypes that are associated with hematopoieticand/or immunological and/or lipid metabolism-related disease statesinclude aberrant proliferation, growth, and migration, anchorageindependent growth, and loss of contact inhibition.

In addition, animal-based hematopoietic and/or immunological and/orlipid metabolism-related disease or disorder systems, such as thosedescribed herein, may be used to identify compounds capable ofameliorating hematopoietic and/or immunological and/or lipidmetabolism-related disease or disorder symptoms. Such animal models maybe used as test substrates for the identification of drugs,pharmaceuticals, therapies, and interventions which may be effective intreating hematopoietic and/or immunological and/or lipidmetabolism-related disorders or diseases. For example, animal models maybe exposed to a compound, suspected of exhibiting an ability tohematopoietic and/or immunological and/or lipid metabolism-relateddisease or disorder symptoms, at a sufficient concentration and for a,time sufficient to elicit such an amelioration of hematopoietic and/orimmunological and/or lipid metabolism-related disease or disordersymptoms in the exposed animals. The response of the animals to theexposure may be monitored by assessing the reversal of disorders orsymptoms associated with hematopoietic and/or immunological and/or lipidmetabolism-related disease.

With regard to intervention, any treatments which reverse any aspect ofhematopoietic and/or immunological and/or lipid metabolism-relateddisease or disorder symptoms should be considered as candidates forhuman hematopoietic and/or immunological and/or lipid metabolism-relateddisease or disorder therapeutic intervention. Dosages of test agents maybe determined by deriving dose-response curves.

Additionally, gene expression patterns may be utilized to assess theability of a compound to ameliorate hematopoietic and/or immunologicaland/or lipid metabolism-related disease symptoms. For example, theexpression pattern of one or more genes may form part of a “geneexpression profile” or “transcriptional profile” which may be then beused in such an assessment. “Gene expression profile” or“transcriptional profile”, as used herein, includes the pattern of mRNAexpression obtained for a given tissue or cell type under a given set ofconditions. Such conditions may include, but are not limited to, cellgrowth, proliferation, differentiation, transformation, tumorigenesis,metastasis, and carcinogen exposure. Gene expression profiles may begenerated, for example, by utilizing a differential display procedure,Northern analysis and/or RT-PCR. In one embodiment, MTP-1 gene sequencesmay be used as probes and/or PCR primers for the generation andcorroboration of such gene expression profiles.

Gene expression profiles may be characterized for known states withinthe cell- and/or animal-based model systems. Subsequently, these knowngene expression profiles may be compared to ascertain the effect a testcompound has to modify such gene expression profiles, and to cause theprofile to more closely resemble that of a more desirable profile.

For example, administration of a compound may cause the gene expressionprofile of a hematopoietic and/or immunological and/or lipidmetabolism-related disease or disorder model system to more closelyresemble the control system. Administration of a compound may,alternatively, cause the gene expression profile of a control system tobegin to mimic a hematopoietic and/or immunological and/or lipidmetabolism-related disease state. Such a compound may, for example, beused in further characterizing the compound of interest, or may be usedin the generation of additional animal models.

B. OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT,HST-4, and/or HST-5

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) predictive medicine (e.g., diagnostic assays,prognostic assays, monitoring clinical trials, and pharmacogenetics);and c) methods of treatment (e.g., therapeutic and prophylactic). Asdescribed herein, an OAT protein of the invention has one or more of thefollowing activities: (i) interaction with an OAT substrate or targetmolecule; (ii) transport of an OAT substrate across a membrane; (iii)interaction with and/or modulation of a second non-OAT protein; (iv)modulation of cellular signaling and/or gene transcription (e.g., eitherdirectly or indirectly); (v) protection of cells and/or tissues fromorganic anions; and/or (vi) modulation, of hormonal responses.

The isolated nucleic acid molecules of the invention can be used, forexample, to express OAT protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect OAT mRNA(e.g., in a biological sample) or a genetic alteration in an OAT gene,and to modulate OAT activity, as described further below. The OATproteins can be used to treat disorders characterized by insufficient orexcessive transport of an OAT substrate or production of OAT inhibitors.In addition, the OAT proteins can be used to screen for naturallyoccurring OAT substrates or target molecules, to screen for drugs orcompounds which modulate OAT activity, as well as to treat disorderscharacterized by insufficient or excessive production of OAT protein orproduction of OAT protein forms which have decreased, aberrant orunwanted activity compared to OAT wild type protein (e.g., anOAT-associated disorder).

Moreover, the anti-OAT antibodies of the invention can be used to detectand isolate OAT proteins, regulate the bioavailability of OAT proteins,and modulate OAT activity.

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) predictive medicine (e.g., diagnostic assays,prognostic assays, monitoring clinical trials, and pharmacogenetics);and c) methods of treatment (e.g., therapeutic and prophylactic). Asdescribed herein, an HST-1 polypeptide of the invention has one or moreof the following activities: (1) maintain sugar homeostasis in a cell,(2) influence insulin and/or glucagon secretion, (3) bind amonosaccharide, e.g., D-glucose, D-fructose, and/or D-galactose, and (4)transport monosaccharides across a cell membrane.

The isolated nucleic acid molecules of the invention can be used, forexample, to express HST-1 polypeptides (e.g., via a recombinantexpression vector in a host cell in gene therapy applications), todetect HST-1 mRNA (e.g., in a biological sample) or a genetic alterationin an HST-1 gene, and to modulate HST-1 activity, as described furtherbelow. The HST-1 polypeptides can be used to treat disorderscharacterized by insufficient or excessive production of an HST-1substrate or production of HST-1 inhibitors. In addition, the HST-1polypeptides can be used to screen for naturally occurring HST-1substrates, to screen for drugs or compounds which modulate HST-1activity, as well as to treat disorders characterized by insufficient orexcessive production of HST-1 polypeptide or production of HST-1polypeptide forms which have decreased, aberrant or unwanted activitycompared to HST-1 wild type polypeptide (e.g., sugar transporterdisorders). Moreover, the anti-HST-1 antibodies of the invention can beused to detect and isolate HST-1 polypeptides, to regulate thebioavailability of HST-1 polypeptides, and modulate HST-1 activity.

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) predictive medicine (e.g., diagnostic assays,prognostic assays, monitoring clinical trials, and pharmacogenetics);and c) methods of treatment (e.g., therapeutic and prophylactic). Asdescribed herein, a TP-2 polypeptide of the invention has one or more ofthe following activities: (1) modulate the import and export ofmolecules, e.g., hormones, ions, cytokines, neurotransmitters,monosaccharides, and metabolites, from cells, 2) modulate intra- orinter-cellular signaling, 3) modulate removal of potentially harmfulcompounds from the cell, or facilitate the compartmentalization of thesemolecules into a sequestered intra-cellular space (e.g., theperoxisome), and 4) modulate transport of biological molecules acrossmembranes, e.g., the plasma membrane, or the membrane of themitochondrion, the peroxisome, the lysosome, the endoplasmic reticulum,the nucleus, or the vacuole.

The isolated nucleic acid molecules of the invention can be used, forexample, to express TP-2 polypeptides (e.g., via a recombinantexpression vector in a host cell in gene therapy applications), todetect TP-2 mRNA (e.g., in a biological sample) or a genetic alterationin a TP-2 gene, and to modulate TP-2 activity, as described furtherbelow. The TP-2 polypeptides can be used to treat disorderscharacterized by insufficient or excessive production of a TP-2substrate or production of TP-2 inhibitors. In addition, the TP-2polypeptides can be used to screen for naturally occurring TP-2substrates, to screen for drugs or compounds which modulate TP-2activity, as well as to treat disorders characterized by insufficient orexcessive production of TP-2 polypeptide or production of TP-2polypeptide forms which have decreased, aberrant or unwanted activitycompared to TP-2 wild type polypeptide (e.g., transporter-associateddisorders). Moreover, the anti-TP-2 antibodies of the invention can beused to detect and isolate TP-2 polypeptides, to regulate thebioavailability of TP-2 polypeptides, and modulate TP-2 activity.

The nucleic acid molecules, proteins, protein homologues, proteinfragments, antibodies, peptides, peptidomimetics, and small moleculesdescribed herein can be used in one or more of the following methods: a)screening assays; b) predictive medicine (e.g., diagnostic assays,prognostic assays, monitoring clinical trials, and pharmacogenetics);and c) methods of treatment (e.g., therapeutic and prophylactic). Asdescribed herein, a PLTR-1 protein of the invention has one or more ofthe following activities: (i) interaction with a PLTR-1 substrate ortarget molecule (e.g., a phospholipid, ATP, or a non-PLTR-1 protein);(ii) transport of a PLTR-l substrate or target molecule (e.g., anaminophospholipid such as phosphatidylserine orphosphatidylethanolamine) from one side of a cellular membrane to theother; (iii) the ability to be phosphorylated or dephosphorylated; (iv)adoption of an E1 conformation or an E2 conformation; (v) conversion ofa PLTR-1 substrate or target molecule to a product (e.g., hydrolysis ofATP); (vi) interaction with a second non-PLTR-1 protein; (vii)modulation of substrate or target molecule location (e.g., modulation ofphospholipid location within a cell and/or location with respect to acellular membrane); (viii) maintenance of aminophospholipid gradients;(ix) modulation of blood coagulation; (x) modulation of intra- orintercellular signaling and/or gene transcription (e.g., either directlyor indirectly); and/or (xi) modulation of cellular proliferation,growth, differentiation, apoptosis, absorption, or secretion.

The isolated nucleic acid molecules of the invention can be used, forexample, to express PLTR-1 protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect PLTR-1mRNA (e.g., in a biological sample) or a genetic alteration in a PLTR-1gene, and to modulate PLTR-1 activity, as described further below. ThePLTR-1 proteins can be used to treat disorders characterized byinsufficient or excessive production or transport of a PLTR-1 substrateor production of PLTR-1 inhibitors, for example, PLTR-1 associateddisorders.

As used interchangeably herein, a “phospholipid transporter associateddisorder” or a “PLTR-1 associated disorder” includes a disorder, diseaseor condition which is caused or characterized by a misregulation (e.g.,downregulation or upregulation) of PLTR-1 activity. PLTR-1 associateddisorders can detrimentally affect cellular functions such as cellularproliferation, growth, differentiation, inter- or intra-cellularcommunication; tissue function, such as cardiac function ormusculoskeletal function; systemic responses in an organism, such asnervous system responses, hormonal responses (e.g., insulin response),or immune responses; and protection of cells from toxic compounds (e.g.,carcinogens, toxins, or mutagens).

Preferred examples of PLTR-1 associated disorders include cardiovascularor cardiac-related disorders. Cardiovascular system disorders in whichthe PLTR-1 molecules of the invention may be directly or indirectlyinvolved include arteriosclerosis, ischemia reperfusion injury,restenosis, arterial inflammation, vascular wall remodeling, ventricularremodeling, rapid ventricular pacing, coronary microembolism,tachycardia, bradycardia, pressure overload, aortic bending, coronaryartery ligation, vascular heart disease, atrial fibrilation, Jervellsyndrome, Lange-Nielsen syndrome, long-QT syndrome, congestive heartfailure, sinus node dysfunction, angina, heart failure, hypertension,atrial fibrillation, atrial flutter, dilated cardiomyopathy, idiopathiccardiomyopathy, myocardial infarction, coronary artery disease, coronaryartery spasm, and arrhythmia. PLTR-1 associated disorders also includedisorders of the musculoskeletal system such as paralysis and muscleweakness, e.g., ataxia, myotonia, and myokymia.

Other examples of PLTR-1 associated disorders include lipid homeostasisdisorders such as atherosclerosis, obesity, diabetes, insulinresistance, hyperlipidemia, hypolipidemia, dyslipidemia,hypercholesterolemia, hypocholesterolemia, triglyceride storage disease,cardiovascular disease, coronary artery disease, hypertension, stroke,overweight, anorexia, cachexia, hyperlipoproteinemia,hypolipoproteinemia, Niemann Pick disease, hypertriglyceridemia,hypotriglyceridemia, pancreatitis, diffuse idiopathic skeletalhyperostosis (DISH), atherogenic lipoprotein phenotype (ALP), epilepsy,liver disease, fatty liver, steatohepatitis, and polycystic ovariansyndrome.

Further examples of PLTR-1 associated disorders include CNS disorderssuch as cognitive and neurodegenerative disorders, examples of whichinclude, but are not limited to, Alzheimer's disease, dementias relatedto Alzheimer's disease (such as Pick's disease), Parkinson's and otherLewy diffuse body diseases, senile dementia, Huntington's disease,Gilles de la Tourette's syndrome, multiple sclerosis, amyotrophiclateral sclerosis, progressive supranuclear palsy, epilepsy, seizuredisorders, and Jakob-Creutzfieldt disease; autonomic function disorderssuch as hypertension and sleep disorders, and neuropsychiatricdisorders, such as depression, schizophrenia, schizoaffective disorder,korsakoff's psychosis, mania, anxiety disorders, or phobic disorders;learning or memory disorders, e.g., amnesia or age-related memory loss,attention deficit disorder, dysthymic disorder, major depressivedisorder, mania, obsessive-compulsive disorder, psychoactive substanceuse disorders, anxiety, phobias, panic disorder, as well as bipolaraffective disorder, e.g., severe bipolar affective (mood) disorder(BP-1), and bipolar affective neurological disorders, e.g., migraine andobesity. Further CNS-related disorders include, for example, thoselisted in the American Psychiatric Association's Diagnostic andStatistical manual of Mental Disorders (DSM), the most current versionof which is incorporated herein by reference in its entirety.

PLTR-1 associated disorders also include cellular proliferation, growth,or differentiation disorders. Cellular proliferation, growth, ordifferentiation disorders include those disorders that affect cellproliferation, growth, or differentiation processes. As used herein, a“cellular proliferation, growth, or differentiation process” is aprocess by which a cell increases in number, size or content, or bywhich a cell develops a specialized set of characteristics which differfrom that of other cells. The PLTR-1 molecules of the present inventionare involved in phospholipid transport mechanisms, which are known to beinvolved in cellular growth, proliferation, and differentiationprocesses. Thus, the PLTR-1 molecules may modulate cellular growth,proliferation, or differentiation, and may play a role in disorderscharacterized by aberrantly regulated growth, proliferation, ordifferentiation. Such disorders include cancer, e.g., carcinoma,sarcoma, or leukemia; tumor angiogenesis and metastasis; skeletaldysplasia; hepatic disorders; and hematopoietic and/ormyeloproliferative disorders.

PLTR-1 associated or related disorders also include hormonal disorders,such as conditions or diseases in which the production and/or regulationof hormones in an organism is aberrant. Examples of such disorders anddiseases include type I and type II diabetes mellitus, pituitarydisorders (e.g., growth disorders), thyroid disorders (e.g.,hypothyroidism or hyperthyroidism), and reproductive or fertilitydisorders (e.g., disorders which affect the organs of the reproductivesystem, e.g., the prostate gland, the uterus, or the vagina; disorderswhich involve an imbalance in the levels of a reproductive hormone in asubject; disorders affecting the ability of a subject to reproduce; anddisorders affecting secondary sex characteristic development, e.g.,adrenal hyperplasia).

PLTR-1 associated or related disorders also include immune disorders,such as autoimmune disorders or immune deficiency disorders, e.g.,congenital X-linked infantile hypogammaglobulinemia, transienthypogammaglobulinemia, common variable immunodeficiency, selective IgAdeficiency, chronic mucocutaneous candidiasis, or severe combinedimmunodeficiency.

PLTR-1 associated or related disorders also include disorders affectingtissues in which PLTR-1 protein is expressed (e.g., vessels).

In addition, the PLTR-1 proteins can be used to screen for naturallyoccurring PLTR-1 substrates, to screen for drugs or compounds whichmodulate PLTR-1 activity, as well as to treat disorders characterized byinsufficient or excessive production of PLTR-1 protein or production ofPLTR-1 protein forms which have decreased, aberrant or unwanted activitycompared to PLTR-1 wild type protein (e.g., a PLTR-1-associateddisorder).

Moreover, the anti-PLTR-1 antibodies of the invention can be used todetect and isolate PLTR-1 proteins, regulate the bioavailability ofPLTR-1 proteins, and modulate PLTR-1 activity.

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) predictive medicine (e.g., diagnostic assays,prognostic assays, monitoring clinical trials, and pharmacogenetics);and c) methods of treatment (e.g., therapeutic and prophylactic). Asdescribed herein, a TFM-2 and/or TFM-3 polypeptide of the invention hasone or more of the following activities: (1) modulate the import andexport of molecules, e.g., hormones, ions, cytokines, neurotransmitters,monocarboxylates, monosaccharides, and metabolites, from cells, 2)modulate intra- or inter-cellular signaling, 3) modulate removal ofpotentially harmful compounds from the cell, or facilitate thecompartmentalization of these molecules into a sequesteredintra-cellular space (e.g., the peroxisome), and 4) modulate transportof biological molecules across membranes, e.g., the plasma membrane, orthe membrane of the mitochondrion, the peroxisome, the lysosome, theendoplasmic reticulum, the nucleus, or the vacuole.

The isolated nucleic acid molecules of the invention can be used, forexample, to express TFM-2 and/or TFM-3 polypeptides (e.g., via arecombinant expression vector in a host cell in gene therapyapplications), to detect TFM-2 and/or TFM-3 mRNA (e.g., in a biologicalsample) or a genetic alteration in a TFM-2 and/or TFM-3 gene, and tomodulate TFM-2 and/or TFM-3 activity, as described further below. TheTFM-2 and/or TFM-3 polypeptides can be used to treat disorderscharacterized by insufficient or excessive production of a TFM-2 and/orTFM-3 substrate or production of TFM-2 and/or TFM-3 inhibitors. Inaddition, the TFM-2 and/or TFM-3 polypeptides can be used to screen fornaturally occurring TFM-2 and/or TFM-3 substrates, to screen for drugsor compounds which modulate TFM-2 and/or TFM-3 activity, as well as totreat disorders characterized by insufficient or excessive production ofTFM-2 and/or TFM-3 polypeptide or production of TFM-2 and/or TFM-3polypeptide forms which have decreased, aberrant or unwanted activitycompared to TFM-2 and/or TFM-3 wild type polypeptide (e.g.,transporter-associated disorders). Moreover, the anti-TFM-2 and/oranti-TFM-3 antibodies of the invention can be used to detect and isolateTFM-2 and/or TFM-3 polypeptides, to regulate the bioavailability ofTFM-2 and/or TFM-3 polypeptides, and modulate TFM-2 and/or TFM-3activity.

The nucleic acid molecules, proteins, protein homologues, antibodies,and modulators described herein can be used in one or more of thefollowing methods: a) screening assays; b) predictive medicine (e.g.,diagnostic assays, prognostic assays, monitoring clinical trials, andpharmacogenetics); and c) methods of treatment (e.g., therapeutic andprophylactic). As described herein, a 67118 or 67067 polypeptide of theinvention has one or more of the following activities: (i) interactionwith a 67118 or 67067 substrate or target molecule (e.g., aphospholipid, ATP, or a non-67118 or 67067 protein); (ii) transport of a67118 or 67067 substrate or target molecule (e.g., an aminophospholipidsuch as phosphatidylserine or phosphatidylethanolamine) from one side ofa cellular membrane to the other; (iii) the ability to be phosphorylatedor dephosphorylated; (iv) adoption of an E1 conformation or an E2conformation; (v) conversion of a 67118 or 67067 substrate or targetmolecule to a product (e.g., hydrolysis of ATP); (vi) interaction with asecond non-67118 or 67067 protein; (vii) modulation of substrate ortarget molecule location (e.g., modulation of phospholipid locationwithin a cell and/or location with respect to a cellular membrane);(viii) maintenance of aminophospholipid gradients; (ix) modulation ofintra- or intercellular signaling and/or gene transcription (e.g.,either directly or indirectly); and/or (x) modulation of cellularproliferation, growth, differentiation, apoptosis, absorption, orsecretion.

The isolated nucleic acid molecules of the invention can be used, forexample, to express 67118 or 67067 polypeptides (e.g., via a recombinantexpression vector in a host cell in gene therapy applications), todetect 67118 or 67067 mRNA (e.g., in a biological sample) or a geneticalteration in a 67118 or 67067 gene, and to modulate 67118 or 67067activity, as described further below. The 67118 or 67067 polypeptidescan be used to treat disorders characterized by insufficient orexcessive production of a 67118 or 67067 substrate or production ortransport of 67118 or 67067 inhibitors, for example, 67118 or 67067associated disorders.

As described herein, a 62092 protein of the invention has one or more ofthe following activities: (i) interaction with a 62092 substrate ortarget molecule (e.g., a nucleotide such as a purine mononucleotide or adinucleoside polyphosphate, or a non-62092 protein); (ii) conversion ofa 62092 substrate or target molecule to a product (e.g., cleavage of anucleoside polyphosphate); (iii) interaction with a second non-62092protein; (iv) sensation of cellular stress signals; (v) regulation ofsubstrate or target molecule availability or activity; (vi) modulationof intra- or intercellular signaling and/or gene transcription (e.g.,either directly or indirectly); and/or (vii) modulation of cellularproliferation, growth, differentiation, and/or apoptosis.

The isolated nucleic acid molecules of the invention can be used, forexample, to express 62092 protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect 62092mRNA (e.g., in a biological sample) or a genetic alteration in a 62092gene, and to modulate 62092 activity, as described further below. The62092 proteins can be used to treat disorders characterized byinsufficient or excessive production of a 62092 substrate or productionof 62092 inhibitors, for example, histidine triad family associateddisorders.

As used interchangeably herein, a “phospholipid transporter associateddisorder” or a “67118 or 67067 associated disorder” includes a disorder,disease or condition which is caused or characterized by a misregulation(e.g., downregulation or upregulation) of 67118 or 67067 activity. 67118or 67067 associated disorders can detrimentally affect cellularfunctions such as cellular proliferation, growth, differentiation,inter- or intra-cellular communication; tissue function, such as cardiacfunction or musculoskeletal function; systemic responses in an organism,such as nervous system responses, hormonal responses (e.g., insulinresponse), or immune responses; and protection of cells from toxiccompounds (e.g., carcinogens, toxins, or mutagens). Examples of 67118 or67067 associated disorders include CNS disorders such as cognitive andneurodegenerative disorders, examples of which include, but are notlimited to, Alzheimer's disease, dementias related to Alzheimer'sdisease (such as Pick's disease), Parkinson's and other Lewy diffusebody diseases, senile dementia, Huntington's disease, Gilles de laTourette's syndrome, multiple sclerosis, amyotrophic lateral sclerosis,progressive supranuclear palsy, epilepsy, seizure disorders, andJakob-Creutzfieldt disease; autonomic function disorders such ashypertension and sleep disorders, and neuropsychiatric disorders, suchas depression, schizophrenia, schizoaffective disorder, korsakoff'spsychosis, mania, anxiety disorders, or phobic disorders; learning ormemory disorders, e.g., amnesia or age-related memory loss, attentiondeficit disorder, dysthymic disorder, major depressive disorder, mania,obsessive-compulsive disorder, psychoactive substance use disorders,anxiety, phobias, panic disorder, as well as bipolar affective disorder,e.g., severe bipolar affective (mood) disorder (BP-1), and bipolaraffective neurological disorders, e.g., migraine and obesity. FurtherCNS-related disorders include, for example, those listed in the AmericanPsychiatric Association's Diagnostic and Statistical manual of MentalDisorders (DSM), the most current version of which is incorporatedherein by reference in its entirety.

Further examples of 67118 or 67067 associated disorders includecardiac-related disorders. Cardiovascular system disorders in which the67118 or 67067 molecules of the invention may be directly or indirectlyinvolved include arteriosclerosis, ischemia reperfusion injury,restenosis, arterial inflammation, vascular wall remodeling, ventricularremodeling, rapid ventricular pacing, coronary microembolism,tachycardia, bradycardia, pressure overload, aortic bending, coronaryartery ligation, vascular heart disease, atrial fibrilation, Jervellsyndrome, Lange-Nielsen syndrome, long-QT syndrome, congestive heartfailure, sinus node dysfunction, angina, heart failure, hypertension,atrial fibrillation, atrial flutter, dilated cardiomyopathy, idiopathiccardiomyopathy, myocardial infarction, coronary artery disease, coronaryartery spasm, and arrhythmia. 67118 or 67067 associated disorders alsoinclude disorders of the musculoskeletal system such as paralysis andmuscle weakness, e.g., ataxia, myotonia, and myokymia.

67118 or 67067 associated disorders also include cellular proliferation,growth, or differentiation disorders. Cellular proliferation, growth, ordifferentiation disorders include those disorders that affect cellproliferation, growth, or differentiation processes. As used herein, a“cellular proliferation, growth, or differentiation process” is aprocess by which a cell increases in number, size or content, or bywhich a cell develops a specialized set of characteristics which differfrom that of other cells. The 67118 or 67067 molecules of the presentinvention are involved in phospholipid transport mechanisms, which areknown to be involved in cellular growth, proliferation, anddifferentiation processes. Thus, the 67118 or 67067 molecules maymodulate cellular growth, proliferation, or differentiation, and mayplay a role in disorders characterized by aberrantly regulated growth,proliferation, or differentiation. Such disorders include cancer, e.g.,carcinoma, sarcoma, or leukemia; tumor angiogenesis and metastasis;skeletal dysplasia; hepatic disorders; and hematopoietic and/ormyeloproliferative disorders.

67118 or 67067 associated or related disorders also include hormonaldisorders, such as conditions or diseases in which the production and/orregulation of hormones in an organism is aberrant. Examples of suchdisorders and diseases include type I and type II diabetes mellitus,pituitary disorders (e.g., growth disorders), thyroid disorders (e.g.,hypothyroidism or hyperthyroidism), and reproductive or fertilitydisorders (e.g., disorders which affect the organs of the reproductivesystem, e.g., the prostate gland, the uterus, or the vagina; disorderswhich involve an imbalance in the levels of a reproductive hormone in asubject; disorders affecting the ability of a subject to reproduce; anddisorders affecting secondary sex characteristic development, e.g.,adrenal hyperplasia).

67118 or 67067 associated or related disorders also include immunedisorders, such as autoimmune disorders or immune deficiency disorders,e.g., congenital X-linked infantile hypogammaglobulinemia, transienthypogammaglobulinemia, common variable immunodeficiency, selective IgAdeficiency, chronic mucocutaneous candidiasis, or severe combinedimmunodeficiency.

67118 or 67067 associated or related disorders also include disordersaffecting tissues in which 67118 or 67067 protein is expressed.

As used interchangeably herein, a “histidine triad family associateddisorder” or a “62092-associated disorder” includes a disorder, diseaseor condition which is caused or characterized by a misregulation (e.g.,downregulation or upregulation) of 62092 activity. 62092 associateddisorders can detrimentally affect cellular functions such as cellularproliferation, growth, differentiation, inter- or intra-cellularcommunication; tissue function, such as cardiac function ormusculoskeletal function; systemic responses in an organism, such asnervous system responses, hormonal responses (e.g., insulin response),or immune responses; and protection of cells from toxic compounds (e.g.,carcinogens, toxins, or mutagens).

In a preferred embodiment, 62092 associated disorders include cellularproliferation, growth, differentiation, or apoptosis disorders. Cellularproliferation, growth, differentiation, or apoptosis disorders includethose disorders that affect cell proliferation, growth, differentiation,or apoptosis processes. As used herein, a “cellular proliferation,growth, differentiation, or apoptosis process” is a process by which acell increases in number, size or content, by which a cell develops aspecialized set of characteristics which differ from that of othercells, or by which a cell undergoes programmed cell death. The 62092molecules of the present invention are involved in nucleotide binding,which are known to be involved in cellular growth, proliferation,differentiation, and apoptosis processes. Thus, the 62092 molecules maymodulate cellular growth, proliferation, differentiation, or apoptosis,and may play a role in disorders characterized by aberrantly regulatedgrowth, proliferation, differentiation, or apoptosis. Such disordersinclude cancer, e.g., carcinoma, sarcoma, or leukemia; tumorangiogenesis and metastasis; skeletal dysplasia; hepatic disorders; andhematopoietic and/or myeloproliferative disorders.

62092 associated disorders also include CNS disorders.

Further examples of 62092 associated disorders include cardiac-relateddisorders, hormonal disorders, and autoimmune disorders or immunedeficiency disorders, as defined herein.

62092 associated or related disorders also include disorders affectingtissues in which 62092 protein is expressed.

In addition, the 67118, 67067, and/or 62092 polypeptides can be used toscreen for naturally occurring 67118, 67067, and/or 62092 substrates, toscreen for drugs or compounds which modulate 67118, 67067, and/or 62092activity, as well as to treat disorders characterized by insufficient orexcessive production of 67118, 67067, and/or 62092 polypeptide orproduction of 67118, 67067, and/or 62092 polypeptide forms which havedecreased, aberrant or unwanted activity compared to 67118, 67067,and/or 62092 wild type polypeptide (e.g., phospholipidtransporter-associated disorders). Moreover, the anti-67118 and/oranti-67067 antibodies of the invention can be used to detect and isolate67118, 67067,. and/or 62092 polypeptides, to regulate thebioavailability of 67118, 67067, and/or 62092 polypeptides, and modulate67118, 67067, and/or 62092 activity.

The nucleic acid molecules, proteins, protein homologues, proteinfragments, antibodies, peptides, peptidomimetics, and small moleculesdescribed herein can be used in one or more of the following methods: a)screening assays; b) predictive medicine (e.g., diagnostic assays,prognostic assays, monitoring clinical trials, and pharmacogenetics);and c) methods of treatment (e.g., therapeutic and prophylactic). Asdescribed herein, a HAAT protein of the invention has one or more of thefollowing activities: (i) interaction with a HAAT substrate or targetmolecule (e.g., an amino acid); (ii) transport of a HAAT substrate ortarget molecule (e.g., an amino acid) from one side of a cellularmembrane to the other; (iii) conversion of a HAAT substrate or targetmolecule to a product (e.g., glucose production); (iv) interaction witha second non-HAAT protein; (v) modulation of substrate or targetmolecule location (e.g., modulation of amino acid location within a celland/or location with respect to a cellular membrane); (vi) maintenanceof amino acid gradients; (vii) modulation of hormone metabolism and/ornerve transmission (e.g., either directly or indirectly); (viii)modulation of cellular proliferation, growth, differentiation, andproduction of metabolic energy; and/or (ix) modulation of amino acidhomeostasis.

The isolated nucleic acid molecules of the invention can be used, forexample, to express HAAT protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect HAAT mRNA(e.g., in a biological sample) or a genetic alteration in a HAAT gene,and to modulate HAAT activity, as described further below. The HAATproteins can be used to treat disorders characterized by insufficient orexcessive production or transport of a HAAT substrate or production ofHAAT inhibitors, for example, HAAT associated disorders.

As used interchangeably herein, a “human amino acid transporterassociated disorder” or a “HAAT associated disorder” includes adisorder, disease or condition which is caused or characterized by amisregulation (e.g., downregulation or upregulation) of HAAT activity.HAAT associated disorders can detrimentally affect cellular functionssuch as protein synthesis, hormone metabolism, nerve transmission,cellular activation, regulation of cell growth, production of metabolicenergy, synthesis of purines and pyrimidines, nitrogen metabolism,and/or biosynthesis of urea. Examples of HAAT associated disordersinclude: retinitis pigmentosa; tumorigenesis; nephrolithiasis; chroniclymphocytic leukemia; neurodegenerative diseases such as epilepsy,ischemia (i.e. hypoxia, stroke), amyotrophic lateral sclerosis; Hatnupdisease; hyperdibasic aminoaciduria; isolated lysinuria;iminoglycinuria; familial protein intolerance; dicarboxylicaminoaciduria; cystinuria; lysinuric protein intolerance; and endotoxicshock.

Further examples of HAAT associated disorders include CNS disorders suchas cognitive and neurodegenerative disorders, examples of which include,but are not limited to, Alzheimer's disease, dementias related toAlzheimer's disease (such as Pick's disease), Parkinson's and other Lewydiffuse body diseases, senile dementia, Huntington's disease, Gilles dela Tourette's syndrome, multiple sclerosis, amyotrophic lateralsclerosis, progressive supranuclear palsy, epilepsy, seizure disorders,and Jakob-Creutzfieldt disease; autonomic function disorders such ashypertension and sleep disorders, and neuropsychiatric disorders, suchas depression, schizophrenia, schizoaffective disorder, korsakoff'spsychosis, mania, anxiety disorders, or phobic disorders; learning ormemory disorders, e.g., amnesia or age-related memory loss, attentiondeficit disorder, dysthymic disorder, major depressive disorder, mania,obsessive-compulsive disorder, psychoactive substance use disorders,anxiety, phobias, panic disorder, as well as bipolar affective disorder,e.g., severe bipolar affective (mood) disorder (BP-1), and bipolaraffective neurological disorders, e.g., migraine and obesity. FurtherCNS-related disorders include, for example, those listed in the AmericanPsychiatric Association's Diagnostic and Statistical manual of MentalDisorders (DSM), the most current version of which is incorporatedherein by reference in its entirety.

As used herein, the term “metabolic disorder” includes a disorder,disease or condition which is caused or characterized by an abnormalmetabolism (i.e., the chemical changes in living cells by which energyis provided for vital processes and activities) in a subject. Metabolicdisorders include diseases, disorders, or conditions associated withaberrant thermogenesis or aberrant adipose cell (e.g., brown or whiteadipose cell) content or function. Metabolic disorders can becharacterized by a misregulation (e.g., downregulation or upregulation)of HAAT activity. Metabolic disorders can detrimentally affect cellularfunctions such as cellular proliferation, growth, differentiation, ormigration, cellular regulation of homeostasis, inter- or intra-cellularcommunication; tissue function, such as liver function, muscle function,or adipocyte function; systemic responses in an organism, such ashormonal responses (e.g., insulin response). Examples of metabolicdisorders include obesity, diabetes, hyperphagia, endocrineabnormalities, triglyceride storage disease, Bardet-Biedl syndrome,Lawrence-Moon syndrome, Prader-Labhart-Willi syndrome, anorexia, andcachexia. Obesity is defined as a body mass index (BMI) of 30 kg/2m ormore (National Institute of Health, Clinical Guidelines on theIdentification, Evaluation, and Treatment of Overweight and Obesity inAdults (1998)). However, the present invention is also intended toinclude a disease, disorder, or condition that is characterized by abody mass index (BMI) of 25 kg/²m or more, 26 kg/²m or more, 27 kg/²m ormore, 28 kg/²m or more, 29 kg/²m or more, 29.5 kg/²m or more, or 29.9kg/²m or more, all of which are typically referred to as overweight(National Institute of Health, Clinical Guidelines on theIdentification, Evaluation, and Treatment of Overweight and Obesity inAdults (1998)).

HAAT associated disorders also include cellular proliferation, growth,or differentiation disorders. Cellular proliferation, growth, ordifferentiation disorders include those disorders that affect cellproliferation, growth, or differentiation processes. As used herein, a“cellular proliferation, growth, or differentiation process” is aprocess by which a cell increases in number, size or content, or bywhich a cell develops a specialized set of characteristics which differfrom that of other cells. The HAAT molecules of the present inventionare involved in amino acid transport mechanisms, which are known to beinvolved in cellular growth, proliferation, and differentiationprocesses. Thus, the HAAT molecules may modulate cellular growth,proliferation, or differentiation, and may play a role in disorderscharacterized by aberrantly regulated growth, proliferation, ordifferentiation. Such disorders include cancer, e.g., carcinoma,sarcoma, or leukemia; tumor angiogenesis and metastasis; skeletaldysplasia; hepatic disorders; and hematopoietic and/ormyeloproliferative disorders.

In addition, the HAAT proteins can be used to screen for naturallyoccurring HAAT substrates, to screen for drugs or compounds whichmodulate HAAT activity, as well as to treat disorders characterized byinsufficient or excessive production of HAAT protein or production ofHAAT protein forms which have decreased, aberrant or unwanted activitycompared to HAAT wild type protein (e.g., a HAAT-associated disorder).

Moreover, the anti-HAAT antibodies of the invention can be used todetect and isolate HAAT proteins, regulate the bioavailability of HAATproteins, and modulate HAAT activity.

The nucleic acid molecules, proteins, protein homologues, antibodies,and modulators described herein can be used in one or more of thefollowing methods: a) screening assays; b) predictive medicine (e.g.,diagnostic assays, prognostic assays, monitoring clinical trials, andpharmacogenetics); and c) methods of treatment (e.g., therapeutic andprophylactic). As described herein, an HST-4 and/or an HST-5 polypeptideof the invention has one or more of the following activities: (1) bind amonosaccharide, e.g., D-glucose, D-fructose, D-galactose, and/ormannose; (2) transport monosaccharides across a cell membrane; (3)influence insulin and/or glucagon secretion; (4) maintain sugarhomeostasis in a cell; and (5) mediate trans-epithelial movement in acell.

The isolated nucleic acid molecules of the invention can be used, forexample, to express HST-4 and/or HST-5 polypeptides (e.g., via arecombinant expression vector in a host cell in gene therapyapplications), to detect HST-4 and/or HST-5 mRNA (e.g., in a biologicalsample) or a genetic alteration in an HST-4 and/or an HST-5 gene, and tomodulate HST-4 and/or HST-5 activity, as described further below. TheHST-4 and/or HST-5 polypeptides, or modulators thereof, can be used totreat disorders characterized by insufficient or excessive production ofan HST-4 and/or an HST-5 substrate or production of HST-4 and/or HST-5inhibitors. In addition, the HST-4 and/or the HST-5 polypeptides can beused to screen for naturally occurring HST-4 and/or HST-5 substrates, toscreen for drugs or compounds which modulate HST-4 and/or HST-5activity, as well as to treat disorders characterized by insufficient orexcessive production of HST-4 and/or HST-5 polypeptide or production ofHST-4 and/or HST-5 polypeptide forms which have decreased, aberrant orunwanted activity compared to HST-4 and/or HST-5 wild type polypeptide(e.g., sugar transporter disorders). Moreover, the anti-HST-4 and/oranti-HST-5 antibodies of the invention can be used to detect and isolateHST-4 and/or HST-5 polypeptides, to regulate the bioavailability ofHST-4 and/or HST-5 polypeptides, and modulate HST-4 and/or HST-5activity.

1. OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT,HST-4 and/or HST-5 Screening Assays:

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)which bind to OAT proteins, PLTR-1 proteins, HAAT proteins have astimulatory or inhibitory effect on, for example, OAT, PLTR-1, HAATexpression or OAT, PLTR-1, HAAT activity, or have a stimulatory orinhibitory effect on, for example, the transport, expression or activityof an OAT substrate or target molecule, a PLTR-1 substrate, a HAATsubstrate.

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)which bind to HST-1 polypeptides, TP-2 polypeptides, TFM-2 polypeptides,TFM-3 polypeptides, 67118, 67067, and/or 62092 polypeptides, HAATpolypeptides, HST-4 and/or HST-5 polypeptides have a stimulatory orinhibitory effect on, for example, HST-1, TP-2, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 expression or HST-1, TP-2, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 activity, or have astimulatory or inhibitory effect on, for example, the expression oractivity of HST-1, TP-2, TFM-2, TFM-3, 67118, 67067, and/or 62092, HAAT,HST-4 and/or HST-5 substrate.

In one embodiment, the invention provides assays for screening candidateor test compounds which are substrates or target molecules of an OATprotein or polypeptide or biologically active portion thereof, an HST-1polypeptide or biologically active portion thereof, a TP-2 polypeptideor biologically active portion thereof, a PLTR-1 protein or polypeptideor biologically active portion thereof, a TFM-2 polypeptide orbiologically active portion thereof, a TFM-3 polypeptide or biologicallyactive portion thereof, a 67118, 67067, and/or 62092 polypeptide orbiologically active portion thereof, a HAAT protein or polypeptide orbiologically active portion thereof, HST-4 and/or HST-5 polypeptide orbiologically active portion thereof. In another embodiment, theinvention provides assays for screening candidate or test compoundswhich bind to or modulate the activity of an OAT protein or polypeptideor biologically active portion thereof. The test compounds of thepresent invention can be obtained using any of the numerous approachesin combinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the‘one-bead one-compound’ library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:45).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example, in: DeWitt et al. (1993) Proc. Natl.Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses an OAT protein or biologically active portion thereof iscontacted with a test compound and the ability of the test compound tomodulate OAT activity is determined. Determining the ability of the testcompound to modulate OAT activity can be accomplished by monitoring, forexample, transport of substrates across membranes and/or levels of genetranscription. The cell, for example, can be of a mammalian origin.

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses an HST-1 polypeptide or biologically active portion thereof iscontacted with a test compound and the ability of the test compound tomodulate HST-1 activity is determined. Determining the ability of thetest compound to modulate HST-1 activity can be accomplished bymonitoring, for example, intracellular or extracellular D-glucose,D-fructose or D-galactose concentration, or insulin or glucagonsecretion. The cell, for example, can be of mammalian origin, e.g., aliver cell, fat cell, muscle cell, or a blood cell, such as anerythrocyte.

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a TP-2 polypeptide or biologically active portion thereof iscontacted with a test compound and the ability of the test compound tomodulate TP-2 activity is determined. Determining the ability of thetest compound to modulate TP-2 activity can be accomplished bymonitoring, for example, intra- or extra-cellular D-glucose, D-fructoseor D-galactose concentration, or insulin or glucagon secretion. Thecell, for example, can be of mammalian origin, e.g., a liver cell, fatcell, muscle cell, or a blood cell, such as an erythrocyte.

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a PLTR-1 protein or biologically active portion thereof iscontacted with a test compound and the ability of the test compound tomodulate PLTR-1 activity is determined. Determining the ability of thetest compound to modulate PLTR-1 activity can be accomplished bymonitoring, for example: (i) interaction of PLTR-1 with a PLTR-1substrate or target molecule (e.g., a phospholipid, ATP, or a non-PLTR-1protein); (ii) transport of a PLTR-1 substrate or target molecule (e.g.,an aminophospholipid such as phosphatidylserine orphosphatidylethanolamine) from one side of a cellular membrane to theother; (iii) the ability of PLTR-1 to be phosphorylated ordephosphorylated; (iv) adoption by PLTR-1 of an E1 conformation or an E2conformation; (v) conversion of a PLTR-1 substrate or target molecule toa product (e.g., hydrolysis of ATP); (vi) interaction of PLTR-1 with asecond non-PLTR-1 protein; (vii) modulation of substrate or targetmolecule location (e.g., modulation of phospholipid location within acell and/or location with respect to a cellular membrane); (viii)maintenance of aminophospholipid gradients; (ix) modulation of bloodcoagulation; (x) modulation of intra- or intercellular signaling and/orgene transcription (e.g., either directly or indirectly); and/or (xi)modulation of cellular proliferation, growth, differentiation,apoptosis, absorption, and/or secretion.

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a TFM-2 and/or TFM-3 polypeptide or biologically activeportion thereof is contacted with a test compound and the ability of thetest compound to modulate TFM-2 and/or TFM-3 activity is determined.Determining the ability of the test compound to modulate TFM-2 and/orTFM-3 activity can be accomplished by monitoring, for example, intra- orextra-cellular lactate, pyruvate, branched chain oxoacid, ketone body,mannose, D-glucose, D-fructose or D-galactose concentration, or insulinor glucagon secretion. The cell, for example, can be of mammalianorigin, e.g., a brain cell, a heart cell, a liver cell, fat cell, musclecell, a tumor cell, or a blood cell, such as an erythrocyte.

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a 67118 and/or 67067 polypeptide or biologically activeportion thereof is contacted with a test compound and the ability of thetest compound to modulate 67118 and/or 67067 activity is determined.Determining the ability of the test compound to modulate 67118 and/or67067 activity can be accomplished by monitoring, for example, (i)interaction of 67118 and/or 67067 with a 67118 and/or 67067 substrate ortarget molecule (e.g., a phospholipid, ATP, or a non-67118 and/or 670672protein); (ii) transport of a 67118 and/or 67067 substrate or targetmolecule (e.g., an aminophospholipid such as phosphatidylserine orphosphatidylethanolamine) from one side of a cellular membrane to theother; (iii) the ability of 67118 and/or 67067 to be phosphorylated ordephosphorylated; (iv) adoption by 67118 and/or 67067 of an E1conformation or an E2 conformation; (v) conversion of a 67118 and/or67067 substrate or target molecule to a product (e.g., hydrolysis ofATP); (vi) interaction of 67118 and/or 67067 with a second non-67118and/or 67067 protein; (vii) modulation of substrate or target moleculelocation (e.g., modulation of phospholipid location within a cell and/orlocation with respect to a cellular membrane); (viii) maintenance ofaminophospholipid gradients; (ix) modulation of intra- or intercellularsignaling and/or gene transcription (e.g., either directly orindirectly); and/or (x) modulation of cellular proliferation, growth,differentiation, apoptosis, absorption, and/or secretion.

In another embodiment, an assay is a cell-based assay in which a cellwhich expresses a 62092 protein or biologically active portion thereofis contacted with a test compound and the ability of the test compoundto modulate 62092 activity is determined. Determining the ability of thetest compound to modulate 62092 activity can be accomplished bymonitoring, for example: (i) interaction with a 62092 substrate ortarget molecule (e.g., a nucleotide such as a purine mononucleotide or adinucleoside polyphosphate, or a non-62092 protein); (ii) conversion ofa 62092 substrate or target molecule to a product (e.g., cleavage of anucleoside polyphosphate); (iii) interaction with a second non-62092protein; (iv) sensation of cellular stress signals; (v) regulation ofsubstrate or target molecule availability or activity; (vi) modulationof intra- or intercellular signaling and/or gene transcription (e.g.,either directly or indirectly); and/or (vii) modulation of cellularproliferation, growth, differentiation, and/or apoptosis.

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a HAAT protein or biologically active portion thereof iscontacted with a test compound and the ability of the test compound tomodulate HAAT activity is determined. Determining the ability of thetest compound to modulate HAAT activity can be accomplished bymonitoring, for example: (i) interaction with a HAAT substrate or targetmolecule (e.g., an amino acid); (ii) transport of a HAAT substrate ortarget molecule (e.g., an amino acid) from one side of a cellularmembrane to the other; (iii) conversion of a HAAT substrate or targetmolecule to a product (e.g., glucose production); (iv) interaction witha second non-HAAT protein; (v) modulation of substrate or targetmolecule location (e.g., modulation of amino acid location within a celland/or location with respect to a cellular membrane); (vi) maintenanceof amino acid gradients; (vii) modulation of hormone metabolism and/ornerve transmission (e.g., either directly or indirectly); (viii)modulation of cellular proliferation, growth, differentiation, andproduction of metabolic energy; and/or (ix) modulation of amino acidhomeostasis.

The activity of the HAAT protein in promoting the uptake of amino acidscan be monitored by expression cloning the HAAT protein in an oocyte. Byincubating the HAAT protein with a ¹⁴C labeled amino acid, the transportof the labeled amino acid into the oocyte by the HAAT protein can bemeasured. Further, the substrate selectivity of the HAAT protein can bemeasured by monitoring the uptake of the ¹⁴C labeled amino acid in thepresence of other non-labeled amino acids which may inhibit the uptakeof the labeled amino acid.

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses an HST-4 and/or an HST-5 polypeptide or biologically activeportion thereof is contacted with a test compound and the ability of thetest compound to modulate HST-4 and/or HST-5 activity is determined.Determining the ability of the test compound to modulate HST-4 and/orHST-5 activity can be accomplished by monitoring, for example,intracellular or extracellular D-glucose, D-fructose, D-galactose,and/or mannose concentration, or insulin or glucagon secretion. Thecell, for example, can be of mammalian origin, e.g., a liver cell, fatcell, muscle cell, or a blood cell, such as an erythrocyte.

The ability of the test compound to modulate binding of a substrate ortarget molecule to OAT can also be determined. The ability of the testcompound to modulate HST-1 binding to a substrate or to bind to HST-1can also be determined. The ability of the test compound to modulateTP-2 binding to a substrate or to bind to TP-2 can also be determined.The ability of the test compound to modulate PLTR-1 binding to asubstrate or to bind to PLTR-1 can also be determined. The ability ofthe test compound to modulate TFM-2 and/or TFM-3 binding to a substrateor to bind to TFM-2 and/or TFM-3 can also be determined. The ability ofthe test compound to modulate 67118, 67067, and/or 62092 binding to asubstrate or to bind to 67118, 67067, and/or 62092 can also bedetermined. The ability of the test compound to modulate HAAT binding toa substrate or to bind to HAAT can also be determined. The ability ofthe test compound to modulate HST-4 and/or HST-5 binding to a substrateor to bind to HST-4 and/or HST-5 can also be determined. Determining theability of the test compound to modulate OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 binding to asubstrate or target molecule can be accomplished, for example, bycoupling the OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 substrate or target molecule with aradioisotope or enzymatic label such that binding of the OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5 substrate or target molecule to OAT can be determined by detectingthe labeled OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5 substrate or target molecule in a complex.Alternatively, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 could be coupled with a radioisotope orenzymatic label to monitor the ability of a test compound to modulateOAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 binding to an OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5 substrate or targetmolecule in a complex. Determining the ability of the test compound tobind OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT,HST-4 and/or HST-5 can be accomplished, for example, by coupling thecompound with a radioisotope or enzymatic label such that binding of thecompound to OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5 can be determined by detecting the labeledcompound in a complex. For example, compounds (e.g., OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5substrates) can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directlyor indirectly, and the radioisotope detected by direct counting ofradioemission or by scintillation counting. Alternatively, compounds canbe enzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of an appropriate substrate to product.

It is also within the scope of this invention to determine the abilityof a compound (e.g., an OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 substrate) to interact with OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 without the labeling of any of the interactants. Forexample, a microphysiometer can be used to detect the interaction of acompound with OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 without the labeling of either thecompound or the OAT. McConnell, H. M. et al. (1992) Science257:1906-1912. As used herein, a “microphysiometer” (e.g., Cytosensor)is an analytical instrument that measures the rate at which a cellacidifies its environment using a light-addressable potentiometricsensor (LAPS). Changes in this acidification rate can be used as anindicator of the interaction between a compound and OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell which expresses OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 with an OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5 target molecule (e.g., an OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5 substrate) and a testcompound and determining the ability of the test compound to modulate(e.g., stimulate or inhibit) the activity (e.g., transport) or cellularlocation of the OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 substrate or target molecule.Determining the ability of the test compound to modulate the activity ofan OAT, HST-1, TP-2, TFM-2, TFM-3, HAAT, HST-4 and/or HST-5 substrate ortarget molecule can be accomplished, for example, by determining theability of the OAT, HST-1, TP-2, TFM-2, TFM-3, HAAT, HST-4 and/or HST-5protein to bind to or interact with the OAT, HST-1, TP-2, TFM-2, TFM-3,HAAT, HST-4 and/or HST-5 substrate or target molecule or by determiningthe cellular localization of the OAT, HST-1, TP-2, TFM-2, TFM-3, HAAT,HST-4 and/or HST-5 substrate or target molecule. Determining the abilityof the test compound to modulate the activity of a PLTR-1 targetmolecule can be accomplished, for example, by determining the ability ofa PLTR-1 protein to bind to or interact with the PLTR-1 target molecule,by determining the cellular location of the target molecule, or bydetermining whether the target molecule (e.g., ATP) has been hydrolyzed.Determining the ability of the test compound to modulate the activity ofa 67118, 67067, and/or 62092 target molecule can be accomplished, forexample, by determining the cellular location of the target molecule, orby determining whether the target molecule (e.g., a 67118 or 67067target molecule such as ATP, or a 62092 target molecule) has beenhydrolyzed.

Determining the ability of the OAT protein, or a biologically activefragment thereof, to bind to or interact with or transport an OATsubstrate or target molecule can be accomplished by one of the methodsdescribed above for determining direct binding. In a preferredembodiment, determining the ability of the OAT protein to bind to orinteract with an OAT substrate or target molecule can be accomplished bydetermining the activity or cellular localization of the substrate ortarget molecule. For example, the activity of the substrate or targetmolecule can be determined by detecting induction of a cellular response(e.g., changes in intracellular substrate concentration), detecting asecondary or indirect activity of the substrate or target molecule,detecting the induction of a reporter gene (comprising atarget-responsive regulatory element operatively linked to a nucleicacid encoding a detectable marker, e.g., luciferase), or detecting atarget-regulated cellular response (i.e., a hormonal response). In otherembodiments, the assays described above are carried out in a cell-freecontext (e.g., in an artificial membrane, vesicle, or micellepreparation).

Determining the ability of the HST-1 polypeptide, or a biologicallyactive fragment thereof, to bind to or interact with an HST-1 targetmolecule can be accomplished by one of the methods described above fordetermining direct binding. In a preferred embodiment, determining theability of the HST-1 polypeptide to bind to or interact with an HST-1target molecule can be accomplished by determining the activity of thetarget molecule. For example, the activity of the target molecule can bedetermined by detecting induction of a cellular second messenger of thetarget (i.e., intracellular Ca²⁺, diacylglycerol, IP₃, and the like),detecting catalytic/enzymatic activity of the target using anappropriate substrate, detecting the induction of a reporter gene(comprising a target-responsive regulatory element operatively linked toa nucleic acid encoding a detectable marker, e.g., luciferase), ordetecting a target-regulated cellular response.

Determining the ability of the TP-2 polypeptide, or a biologicallyactive fragment thereof, to bind to or interact with a TP-2 targetmolecule can be accomplished by one of the methods described above fordetermining direct binding. In a preferred embodiment, determining theability of the TP-2 polypeptide to bind to or interact with a TP-2target molecule can be accomplished by determining the activity of thetarget molecule. For example, the activity of the target molecule can bedetermined by detecting induction of a cellular second messenger of thetarget (i.e., intra-cellular Ca²⁺, diacylglycerol, IP₃, and the like),detecting catalytic/enzymatic activity of the target using anappropriate substrate, detecting the induction of a reporter gene(comprising a target-responsive regulatory element operatively linked toa nucleic acid encoding a detectable marker, e.g., luciferase), ordetecting a target-regulated cellular response.

Determining the ability of the PLTR-1 protein, or a biologically activefragment thereof, to bind to or interact with a PLTR-1 target moleculecan be accomplished by one of the methods described above fordetermining direct binding. In a preferred embodiment, determining theability of the PLTR-1 protein to bind to or interact with a PLTR-1target molecule can be accomplished by determining the activity of thetarget molecule. For example, the activity of the target molecule can bedetermined by detecting the cellular location of target molecule,detecting catalytic/enzymatic activity of the target molecule upon anappropriate substrate, detecting induction of a metabolite of the targetmolecule (e.g., detecting the products of ATP hydrolysis) detecting theinduction of a reporter gene (comprising a target-responsive regulatoryelement operatively linked to a nucleic acid encoding a detectablemarker, e.g., luciferase), or detecting a target-regulated cellularresponse (i.e., cell growth or differentiation).

Determining the ability of the TFM-2 and/or TFM-3 polypeptide, or abiologically active fragment thereof, to bind to or interact with aTFM-2 and/or TFM-3 target molecule can be accomplished by one of themethods described above for determining direct binding. In a preferredembodiment, determining the ability of the TFM-2 and/or TFM-3polypeptide to bind to or interact with a TFM-2 and/or TFM-3 targetmolecule can be accomplished by determining the activity of the targetmolecule. For example, the activity of the target molecule can bedetermined by detecting induction of a cellular second messenger of thetarget (i.e., intra-cellular Ca²⁺, diacylglycerol, IP₃, and the like),detecting catalytic/enzymatic activity of the target using anappropriate substrate, detecting the induction of a reporter gene(comprising a target-responsive regulatory element operatively linked toa nucleic acid encoding a detectable marker, e.g., luciferase), ordetecting a target-regulated cellular response.

Determining the ability of the 67118, 67067, and/or 62092 polypeptide,or a biologically active fragment thereof, to bind to or interact with a67118, 67067, and/or 62092 target molecule can be accomplished by one ofthe methods described above for determining direct binding. In apreferred embodiment, determining the ability of the 67118, 67067,and/or 62092 polypeptide to bind to or interact with a 67118, 67067,and/or 62092 target molecule can be accomplished by determining theactivity of the target molecule. For example, the activity of the targetmolecule can be determined by detecting the cellular location of targetmolecule, detecting catalytic/enzymatic activity of the target moleculeupon an appropriate substrate, detecting induction of a metabolite ofthe target molecule (e.g., detecting the products of ATP hydrolysis)detecting the induction of a reporter gene (comprising atarget-responsive regulatory element operatively linked to a nucleicacid encoding a detectable marker, e.g., luciferase), or detecting atarget-regulated cellular response (i.e., cell growth ordifferentiation).

Determining the ability of the HST-4 and/or the HST-5 polypeptide, or abiologically active fragment thereof, to bind to or interact with anHST-4 and/or an HST-5 target molecule can be accomplished by one of themethods described above for determining direct binding. In a preferredembodiment, determining the ability of the HST-4 and/or the HST-5polypeptide to bind to or interact with an HST-4 and/or an HST-5 targetmolecule can be accomplished by determining the activity of the targetmolecule. For example, the activity of the target molecule can bedetermined by detecting induction of a cellular second messenger of thetarget, detecting catalytic/enzymatic activity of the target using anappropriate substrate, detecting the induction of a reporter gene(comprising a target-responsive regulatory element operatively linked toa nucleic acid encoding a detectable marker, e.g., luciferase), ordetecting a target-regulated cellular response.

In yet another embodiment, an assay of the present invention is acell-free assay in which an OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5 protein or biologicallyactive portion (e.g., a portion which possesses the ability to transportor interact with a substrate or target molecule) thereof is contactedwith a test compound and the ability of the test compound to bind to theOAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 protein or biologically active portion thereof isdetermined. Preferred biologically active portions of the OAT proteinsto be used in assays of the present invention include fragments whichparticipate in interactions with non-OAT molecules, e.g., fragments withhigh surface probability scores (see, for example, FIGS. 4 and 5).Preferred biologically active portions of the HST-1 polypeptides to beused in assays of the present invention include fragments whichparticipate in interactions with non-HST-1 molecules, e.g., fragmentswith high surface probability scores (see, for example, FIG. 7).Preferred biologically active portions of the TP-2 polypeptides to beused in assays of the present invention include fragments whichparticipate in interactions with non-TP-2 molecules, e.g., fragmentswith high surface probability scores (see, for example, FIG. 11).Preferred biologically active portions of the PLTR-1 proteins to be usedin assays of the present invention include fragments which participatein interactions with non-PLTR-1 molecules, e.g., fragments with highsurface probability scores (see, for example, FIG. 15). Preferredbiologically active portions of the TFM-2 and/or TFM-3 polypeptides tobe used in assays of the present invention include fragments whichparticipate in interactions with non-TFM-2 and/or non-TFM-3 molecules,e.g., fragments with high surface probability scores (see, for example,FIGS. 16 and 18). Preferred biologically active portions of the 67118,67067, and/or 62092 polypeptides to be used in assays of the presentinvention include fragments which participate in interactions withnon-67118, non-67067, and/or non-62092 molecules, e.g., fragments withhigh surface probability scores (see, for example, FIGS. 20, 22, and24). Preferred biologically active portions of the HAAT proteins to beused in assays of the present invention include fragments whichparticipate in interactions with non-HAAT molecules. Preferredbiologically active portions of the HST-4 and/or the HST-5 polypeptidesto be used in assays of the present invention include fragments whichparticipate in interactions with non-HST-4 and/or non-HST-5 molecules,e.g., fragments with high surface probability scores (see, for example,FIGS. 29 and 30). Binding of the test compound to the OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5protein can be determined either directly or indirectly as describedabove. In a preferred embodiment, the assay includes contacting the OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 protein or biologically active portion thereof with a knowncompound which binds OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with an OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5protein, wherein determining the ability of the test compound tointeract with an OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 protein comprises determining theability of the test compound to preferentially bind to OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 orbiologically active portion thereof as compared to the known compound.

In another embodiment, the assay is a cell-free assay in which a TFM-2and/or TFM-3 polypeptide, or biologically active portion thereof, iscontacted with a test compound and the ability of the test compound tomodulate the intrinsic fluorescence of the TFM-2 and/or TFM-3polypeptide, or biologically active portion thereof, is monitored. It iscommon for a molecule's intrinsic fluorescence to change when bindingoccurs with or near fluorescent aminoacids (e.g., tryptophan andtyrosine).

In another embodiment, the assay is a cell-free assay in which an OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 protein or biologically active portion thereof is contactedwith a test compound and the ability of the test compound to modulate(e.g., stimulate or inhibit) the activity of the OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5protein or biologically active portion thereof is determined.Determining the ability of the test compound to modulate the activity ofan OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT,HST-4 and/or HST-5 protein can be accomplished, for example, bydetermining the ability of the OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5 protein to bind to an OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 substrate or target molecule by one of the methodsdescribed above for determining direct binding. Determining the abilityof the OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5 protein to bind to an OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 substrate ortarget molecule can also be accomplished using a technology such asreal-time Biomolecular Interaction Analysis (BIA). Sjolander, S. andUrbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995)Curr. Opin. Struct. Biol. 5:699-705. As used herein, “BIA” is atechnology for studying biospecific interactions in real time, withoutlabeling any of the interactants (e.g., BIAcore). Changes in the opticalphenomenon of surface plasmon resonance (SPR) can be used as anindication of real-time reactions between biological molecules.

In an alternative embodiment, determining the ability of the testcompound to modulate the activity of an OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 protein can beaccomplished by determining the ability of the OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 protein tofurther modulate the activity of a downstream effector of an OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5 target molecule. For example, the activity of the effectormolecule on an appropriate target can be determined or the binding ofthe effector to an appropriate target can be determined as previouslydescribed.

In yet another embodiment, the cell-free assay involves contacting anOAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 protein or biologically active portion thereof with a knowncompound which binds to or is transported by the OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5protein to form an assay mixture, contacting the assay mixture with atest compound, and determining the ability of the test compound tointeract with the OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 protein, wherein determining the abilityof the test compound to interact with the OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 proteincomprises determining the ability of the OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 protein topreferentially bind to, transport, or modulate the activity of an OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 substrate or target molecule.

The cell-free assays of the present invention are amenable to use ofboth soluble and/or membrane-bound forms of isolated proteins (e.g.,OAT, PLTR-1, 67118, 67067, and/or 62092 or HAAT proteins or biologicallyactive portions thereof). In the case of cell-free assays in which amembrane-bound form of an isolated protein is used it may be desirableto utilize a solubilizing agent such that the membrane-bound form of theisolated protein is maintained in solution. Examples of suchsolubilizing agents include non-ionic detergents such asn-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either OAT or its substrateor target molecule to facilitate separation of complexed fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound to anOAT protein, or interaction of an OAT protein with a substrate or targetmolecule in the presence and absence of a candidate compound, can beaccomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtiter plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided which adds a domain that allows one or both of the proteins tobe bound to a matrix. For example, glutathione-S-transferase/OAT fusionproteins or glutathione-S-transferase/target fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized micrometer plates, which are thencombined with the test compound or the test compound and either thenon-adsorbed target protein or OAT protein, and the mixture incubatedunder conditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of OATbinding or activity determined using standard techniques.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either HST-1 or its targetmolecule, either TP-2 or its target molecule, either PLTR-1 or itstarget molecule, either TFM-2 or its target molecule, either TFM-3 orits target molecule, either 67118, 67067, and/or 62092 or their targetmolecules, HAAT or its target molecule, HST-4 or its target molecule,and/or HST-5 or its target molecule, to facilitate separation ofcomplexed from uncomplexed forms of one or both of the proteins, as wellas to accommodate automation of the assay. Binding of a test compound toa HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 polypeptide, or interaction of a HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 polypeptidewith a target molecule in the presence and absence of a candidatecompound, can be accomplished in any vessel suitable for containing thereactants. Examples of such vessels include microtiter plates, testtubes, and micro-centrifuge tubes. In one embodiment, a fusion proteincan be provided which adds a domain that allows one or both of theproteins to be bound to a matrix. For example,glutathione-S-transferase/HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized micrometer plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 polypeptide, and the mixture incubatedunder conditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrometer plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level ofHST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either an OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 protein or an OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5 substrate or targetmolecule can be immobilized utilizing conjugation of biotin andstreptavidin. Biotinylated OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5 protein, substrates ortarget molecules can be prepared from biotin-NHS (N-hydroxy-succinimide)using techniques known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,antibodies reactive with OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 protein, substrates or targetmolecules but which do not interfere with binding of the OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5 protein to its substrate, or target molecule can be derivatized tothe wells of the plate, and unbound target or OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 proteintrapped in the wells by antibody conjugation. Methods for detecting suchcomplexes, in addition to those described above for the GST-immobilizedcomplexes, include immunodetection of complexes using antibodiesreactive with the OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 protein or target molecule, as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 protein or target molecule.

In another embodiment, modulators of OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 expression areidentified in a method wherein a cell is contacted with a candidatecompound and the expression of OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5 mRNA or protein in thecell is determined. The level of expression of OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 mRNA orprotein in the presence of the candidate compound is compared to thelevel of expression of OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 mRNA or protein in the absence ofthe candidate compound. The candidate compound can then be identified asa modulator of OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 expression based on this comparison. Forexample, when expression of OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5 mRNA or protein is greater(statistically significantly greater) in the presence of the candidatecompound than in its absence, the candidate compound is identified as astimulator of OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 mRNA or protein expression.Alternatively, when expression of OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 mRNA or protein isless (statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 mRNA or protein expression. The level ofOAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 mRNA or protein expression in the cells can be determinedby methods described herein for detecting OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 mRNA orprotein.

In yet another aspect of the invention, the OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 proteins canbe used as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with OAT (“OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5-binding proteins” or“OAT-bp”, “HST-1-bp”, “TP-2-bp”, “PLTR-1-bp”, “TFM-2-bp”, “TFM-3-bp”,“67118-bp”, “67067-bp”, “62092-bp”, “HAAT-bp”, “HST-4-bp” and/or“HST-5-bp”) and are involved in OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5 activity. Such OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5-binding proteins are also likely to be involved in the propagationof signals by the OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 proteins or OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 targets as,for example, downstream elements of an OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5-mediated signalingpathway. Alternatively, such OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5-binding proteins may beOAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for an OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5 protein is fused to a gene encoding the DNA binding domain of aknown transcription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming an OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5-dependent complex, the DNA-binding and activation domainsof the transcription factor are brought into close proximity. Thisproximity allows transcription of a reporter gene (e.g., LacZ) which isoperably linked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 protein.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell free assay, and the abilityof the agent to modulate the activity of an OAT protein can be confirmedin vivo, e.g., in an animal such as an animal model for organic anionsensitivity or an animal model with dysregulated organic aniontransport.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell free assay, and the abilityof the agent to modulate the activity of an HST-1 polypeptide can beconfirmed in vivo, e.g., in an animal such as an animal model forobesity, or diabetes. Examples of animals that can be used include thetransgenic mouse described in U.S. Pat. No. 5,932,779 that contains amutation in an endogenous melanocortin-4-receptor (MC4-R) gene; animalshaving mutations which lead to syndromes that include obesity symptoms(described in, for example, Friedman, J. M. et al. (1991) Mamm. Gen.1:130-144; Friedman, J. M. and Liebel, R. L. (1992) Cell 69:217-220;Bray, G. A. (1992) Prog. Brain Res. 93:333-341; and Bray, G. A. (1989)Amer. J. Clin. Nutr. 5:891-902); the animals described in Stubdal H. etal. (2000) Mol. Cell Biol. 20(3):878-82 (the mouse tubby phenotypecharacterized by maturity-onset obesity); the animals described inAbadie J. M. et al. Lipids (2000) 35(6):613-20 (the obese Zucker rat(ZR), a genetic model of human youth-onset obesity and type 2 diabetesmellitus); the animals described in Shaughnessy S. et al. (2000)Diabetes 49(6):904-11 (mice null for the adipocyte fatty acid bindingprotein); or the animals described in Loskutoff D. J. et al. (2000) Ann.N. Y Acad. Sci. 902:272-81 (the fat mouse). Other examples of animalsthat may be used include non-recombinant, non-genetic animal models ofobesity such as, for example, rabbit, mouse, or rat models in which theanimal has been exposed to either prolonged cold or long-termover-eating, thereby, inducing hypertrophy of BAT and increasing BATthermogenesis (Himms-Hagen, J. (1990), supra). Additionally, animalscreated by ablation of BAT through use of targeted expression of a toxingene (Lowell, B. et al. (1993) Nature 366:740-742) may be used.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell free assay, and the abilityof the agent to modulate the activity of a TP-2 polypeptide can beconfirmed in vivo, e.g., in an animal such as an animal model forcellular transformation and/or tumorigenesis.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell-free assay, and the abilityof the agent to modulate the activity of a PLTR-1 protein can beconfirmed in vivo, e.g., in an animal such as an animal model forcellular transformation and/or tumorigenesis.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell free assay, and the abilityof the agent to modulate the activity of a TFM-2 and/or TFM-3polypeptide can be confirmed in vivo, e.g., in an animal such as ananimal model for cellular transformation and/or tumorigenesis, an animalmodel for obesity, or an animal model for a deficiency in sugartransport. Examples of animals that can be used include animals havingmutations which lead to syndromes that include obesity symptoms(described in, for example, Friedman, J. M. et al. (1991) Mamm. Gen.1:130-144; Friedman, J. M. and Liebel, R. L. (1992) Cell 69:217-220;Bray, G. A. (1992) Prog. Brain Res. 93:333-341; and Bray, G. A. (1989)Amer. J. Clin. Nutr. 5:891-902); the animals described in Stubdal H. etal. (2000) Mol. Cell Biol. 20(3):878-82 (the mouse tubby phenotypecharacterized by maturity-onset obesity); the animals described inAbadie J. M. et al. Lipids (2000) 35(6):613-20 (the obese Zucker rat(ZR), a genetic model of human youth-onset obesity and type 2 diabetesmellitus); the animals described in Shaughnessy S. et al. (2000)Diabetes 49(6):904-11 (mice null for the adipocyte fatty acid bindingprotein); or the animals described in Loskutoff D. J. et al. (2000) Ann.N. Y. Acad. Sci. 902:272-81 (the fat mouse).

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell free assay, and the abilityof the agent to modulate the activity of a 67118, 67067, and/or 62092polypeptide can be confirmed in vivo, e.g., in an animal such as ananimal model for cellular transformation and/or tumorigenesis, such asanimal models for colon cancer or lung cancer. Animal based models forstudying tumorigenesis in vivo are well known in the art (reviewed inAnimal Models of Cancer Predisposition Syndromes, Hiai, H and Hino, O(eds.) 1999, Progress in Experimental Tumor Research, Vol. 35; Clarke AR Carcinogenesis (2000) 21:435-41) and include, for example,carcinogen-induced tumors (Rithidech, K et al. Mutat. Res. (1999)428:33-39; Miller, M. L. et al. Environ Mol Mutagen (2000) 35:319-327),injection and/or transplantation of tumor cells into an animal, as wellas animals bearing mutations in growth regulatory genes, for example,oncogenes (e.g., ras) (Arbeit, J M et al. Am J Pathol (1993)142:1187-1197; Sinn, E et al. Cell (1987) 49:465-475; Thorgeirsson, S Set al. Toxicol Lett (2000) 112-113:553-555) and tumor suppressor genes(e.g., p 53) (Vooijs, M et al. Oncogene (1999) 18:5293-5303; Clark A RCancer Metast Rev (1995) 14:125-148; Kumar, T R et al. J Intern Med(1995) 238:233-238; Donehower, L A et al. (1992) Nature 356215-221).Furthermore, experimental model systems are available for the study of,for example, ovarian cancer (Hamilton, T C et al. Semin Oncol (1984)11:285-298; Rahman, N A et al. Mol Cell Endocrinol (1998) 145:167-174;Beamer, W G et al. Toxicol Pathol (1998) 26:704-710), gastric cancer(Thompson, J et al. (2000) Int. J. Cancer 86:863-869; Fodde, R et al.Cytogenet Cell Genet (1999) 86:105-111), breast cancer (Li, M et al.Oncogene (2000) 19:1010-1019; Green, J E et al. Oncogene (2000)19:1020-1027), melanoma (Satyamoorthy, K et al. Cancer Metast Rev (1999)18:401-405), and prostate cancer (Shirai, T et al. Mutat. Res. (2000)462:219-226; Bostwick, D G et al. Prostate (2000) 43:286-294).

In yet another aspect of the invention, the HAAT proteins can be used as“bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g.,U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura etal. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and Brent WO94/10300) to identify other proteins which bind to orinteract with HAAT (“HAAT-binding proteins” or “HAAT-bp”) and areinvolved in HAAT activity.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell free assay, and the abilityof the agent to modulate the activity of an HST-4 and/or an HST-5polypeptide can be confirmed in vivo, e.g., in animal models forobesity, anorexia, type-1 diabetes, type-2 diabetes, hypoglycemia,glycogen storage disease (Von Gierke disease), type I glycogenosis,bipolar disorder, seasonal affective disorder, cluster B personalitydisorders, cellular transformation, and/or tumorigenesis. Examples ofanimal models which may be used include animals having mutations whichlead to syndromes that include obesity symptoms (described in, forexample, Friedman, J. M. et al. (1991) Mamm. Gen. 1:130-144; Friedman,J. M. and Liebel, R. L. (1992) Cell 69:217-220; Bray, G. A. (1992) Prog.Brain Res. 93:333-341; and Bray, G. A. (1989) Amer. J. Clin. Nutr.5:891-902); the animals described in Stubdal H. et al. (2000) Mol. CellBiol. 20(3):878-82 (the mouse tubby phenotype characterized bymaturity-onset obesity); the animals described in Abadie J. M. et al.Lipids (2000) 35(6):613-20 (the obese Zucker rat (ZR), a genetic modelof human youth-onset obesity and type 2 diabetes mellitus); the animalsdescribed in Shaughnessy S. et al. (2000) Diabetes 49(6):904-11 (micenull for the adipocyte fatty acid binding protein); or the animalsdescribed in Loskutoff D. J. et al. (2000) Ann. N. Y. Acad. Sci.902:272-81 (the fat mouse).

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., an OAT substrate, an OAT target molecule, an OATmodulating agent, an antisense OAT nucleic acid molecule, anOAT-specific antibody, or an OAT binding partner, an HST-1 modulatingagent, an antisense HST-1 nucleic acid molecule, an HST-1-specificantibody, or an HST-1-binding partner, a TP-2 modulating agent, anantisense TP-2 nucleic acid molecule, a TP-2-specific antibody, or aTP-2-binding partner, a PLTR-1 modulating agent, an antisense PLTR-1nucleic acid molecule, a PLTR-1-specific antibody, or a PLTR-1 bindingpartner, a TFM-2 and/or TFM-3 modulating agent, an anti sense TFM-2and/or TFM-3 nucleic acid molecule, a TFM-2 and/or TFM-3-specificantibody, or a TFM-2 and/or TFM-3-binding partner, a 67118, 67067,and/or 62092 modulating agent, an antisense 67118, 67067, and/or 62092nucleic acid molecule, a 67118, 67067, and/or 62092-specific antibody,or a 67118, 67067, and/or 62092-binding partner, a HAAT modulatingagent, an antisense HAAT nucleic acid molecule, a HAAT-specificantibody, or a HAAT binding partner, an HST-4 and/or an HST-5 modulatingagent, an antisense HST-4 and/or HST-5 nucleic acid molecules, an HST-4-and/or an HST-5-specific antibody, or an HST-4- and/or an HST-5-bindingpartner) can be used in an animal model to determine the efficacy,toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal model to determine the mechanism of action of such an agent.Furthermore, this invention pertains to uses of novel agents identifiedby the above-described screening assays for treatments as describedherein.

C. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

1. Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. This process is called chromosome mapping. Accordingly,portions or fragments of the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or the HST-5 nucleotidesequences, described herein, can be used to map the location of theMTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or the HST-5 genes on a chromosome. The mapping of theMTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or the HST-5 sequences to chromosomes is an importantfirst step in correlating these sequences with genes associated withdisease.

Briefly, MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 genes can be mapped to chromosomes bypreparing PCR primers (preferably 15-25 bp in length) from the MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or the HST-5 nucleotide sequences. Computer analysis of the MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or the HST-5 sequences can be used to predict primers that do notspan more than one exon in the genomic DNA, thus complicating theamplification process. These primers can then be used for PCR screeningof somatic cell hybrids containing individual human chromosomes. Onlythose hybrids containing the human gene corresponding to the MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or the HST-5 sequences will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow, because they lack a particular enzyme, but humancells can, the one human chromosome that contains the gene encoding theneeded enzyme, will be retained. By using various media, panels ofhybrid cell lines can be established. Each cell line in a panel containseither a single human chromosome or a small number of human chromosomes,and a full set of mouse chromosomes, allowing easy mapping of individualgenes to specific human chromosomes. (D'Eustachio P. et al. (1983)Science 220:919-924). Somatic cell hybrids containing only fragments ofhuman chromosomes can also be produced by using human chromosomes withtranslocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or the HST-5 nucleotide sequences to design oligonucleotide primers,sublocalization can be achieved with panels of fragments from specificchromosomes. Other mapping strategies which can similarly be used to mapan MTP-1, an OAT, an HST-1, a TP-2, a PLTR-1, a TFM-2, a TFM-3, a 67118,a 67067, a 62092, a HAAT, an HST-4 and/or an HST-5 sequence to itschromosome include in situ hybridization (described in Fan, Y. et al.(1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening withlabeled flow-sorted chromosomes, and pre-selection by hybridization tochromosome specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical such ascolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see Verma et al., Human Chromosomes: A Manual ofBasic Techniques (Pergamon Press, New York 1988).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship between agene and a disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, for example, Egeland, J. et al. (1987)Nature, 325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or theHST-5 gene, can be determined. If a mutation is observed in some or allof the affected individuals but not in any unaffected individuals, thenthe mutation is likely to be the causative agent of the particulardisease. Comparison of affected and unaffected individuals generallyinvolves first looking for structural alterations in the chromosomes,such as deletions or translocations that are visible from chromosomespreads or detectable using PCR based on that DNA sequence. Ultimately,complete sequencing of genes from several individuals can be performedto confirm the presence of a mutation and to distinguish mutations frompolymorphisms.

2. Tissue Typing

The MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or the HST-5 sequences of the present invention can alsobe used to identify individuals from minute biological samples. TheUnited States military, for example, is considering the use ofrestriction fragment length polymorphism (RFLP) for identification ofits personnel. In this technique, an individual's genomic DNA isdigested with one or more restriction enzymes, and probed on a Southernblot to yield unique bands for identification. This method does notsuffer from the current limitations of “Dog Tags” which can be lost,switched, or stolen, making positive identification difficult. Thesequences of the present invention are useful as additional DNA markersfor RFLP (described in U.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or the HST-5 nucleotide sequencesdescribed herein can be used to prepare two PCR primers from the 5′ and3′ ends of the sequences. These primers can then be used to amplify anindividual's DNA and subsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or the HST-5 nucleotide sequences of the inventionuniquely represent portions of the human genome. Allelic variationoccurs to some degree in the coding regions of these sequences, and to agreater degree in the noncoding regions. It is estimated that allelicvariation between individual humans occurs with a frequency of aboutonce per each 500 bases. Each of the sequences described herein can, tosome degree, be used as a standard against which DNA from an individualcan be compared for identification purposes. Because greater numbers ofpolymorphisms occur in the noncoding regions, fewer sequences arenecessary to differentiate individuals. The noncoding sequences of SEQID NO:1, 4, 7, 12, 15, 19, 27, 30, 33, 36, 39, 51, 54 or 57 cancomfortably provide positive individual identification with a panel ofperhaps 10 to 1,000 primers which each yield a noncoding amplifiedsequence of 100 bases. If predicted coding sequences, such as those inSEQ ID NO:3, 6, 9, 14, 17, 21, 29, 32, 35, 38, 41, 53, 56, or 59 areused, a more appropriate number of primers for positive individualidentification would be 500-2,000.

If a panel of reagents from MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 nucleotidesequences described herein is used to generate a unique identificationdatabase for an individual, those same reagents can later be used toidentify tissue from that individual. Using the unique identificationdatabase, positive identification of the individual, living or dead, canbe made from extremely small tissue samples.

3. Use of MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and HST-5 Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NO:1, 4, 7, 12, 15, 19, 27, 30,33, 36, 39, 51, 54 or 57 are particularly appropriate for this use asgreater numbers of polymorphisms occur in the noncoding regions, makingit easier to differentiate individuals using this technique. Examples ofpolynucleotide reagents include the MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or the HST-5nucleotide sequences or portions thereof, e.g., fragments derived fromthe noncoding regions of SEQ ID NO:1, 4, 7, 12, 15, 19, 27, 30, 33, 36,39, 51, 54 or 57 having a length of at least 20 bases, preferably atleast 30 bases.

The MTP-1 nucleotide sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue, e.g., thymus or brain tissue. This can bevery useful in cases where a forensic pathologist is presented with atissue of unknown origin. Panels of such MTP-1 probes can be used toidentify tissue by species and/or by organ type.

The OAT nucleotide sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue, e.g., an OAT-expressing tissue. This can bevery useful in cases where a forensic pathologist is presented with atissue of unknown origin. Panels of such OAT probes can be used toidentify tissue by species and/or by organ type.

The PLTR-1 or HAAT nucleotide sequences described herein can further beused to provide polynucleotide reagents, e.g., labeled or labelableprobes which can be used in, for example, an in situ hybridizationtechnique, to identify a specific tissue, e.g., a tissue which expressesPLTR-1 or a tissue which expresses HAAT. This can be very useful incases where a forensic pathologist is presented with a tissue of unknownorigin. Panels of such PLTR-1 or HAAT probes can be used to identifytissue by species and/or by organ type.

The HST-1, TP-2, TFM-2, TFM-3, 67118, 67067, 62092, HST-4 and/or theHST-5 nucleotide sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue, e.g., brain tissue. This can be very usefulin cases where a forensic pathologist is presented with a tissue ofunknown origin. Panels of such HST-1, TP-2, TFM-2, TFM-3, 67118, 67067,62092, HST-4 and/or HST-5 probes can be used to identify tissue byspecies and/or by organ type.

In a similar fashion, these reagents, e.g., MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5primers or probes can be used to screen tissue culture for contamination(i.e. screen for the presence of a mixture of different types of cellsin a culture).

D. Predictive Medicine:

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual prophylactically. Accordingly, one aspect of the presentinvention relates to diagnostic assays for determining MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 polypeptide and/or nucleic acid expression as well asMTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5 activity, in the context of a biological sample(e.g., blood, serum, cells, tissue) to thereby determine whether anindividual is afflicted with a disease or disorder, or is at risk ofdeveloping a disorder, associated with aberrant or unwanted MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 expression or activity. The invention also provides forprognostic (or predictive) assays for determining whether an individualis at risk of developing a disorder associated with MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5 polypeptides, nucleic acid expression or activity. For example,mutations in an MTP-1, OAT, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or an HST-5 gene can be assayed in a biologicalsample. Such assays can be used for prognostic or predictive purpose tothereby prophylactically treat an individual prior to the onset of adisorder characterized by or associated with MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5polypeptides, nucleic acid expression or activity.

Another aspect of the invention pertains to monitoring the influence ofagents (e.g., drugs, compounds) on the expression or activity of MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 in clinical trials.

These and other agents are described in further detail in the followingsections.

1. Regarding MTP-1

The present invention encompasses methods for diagnostic and prognosticevaluation of hematopoietic and/or immunological and/or lipidmetabolism-related disorders or diseases, e.g., atherogenesis,including, but not limited to colon cancer and lung cancer, and for theidentification of subjects exhibiting a predisposition to suchconditions.

An exemplary method for detecting the presence or absence of MTP-1protein or nucleic acid in a biological sample involves obtaining abiological sample from a test subject and contacting the biologicalsample with a compound or an agent capable of detecting MTP-1 protein ornucleic acid (e.g., mRNA, or genomic DNA) that encodes MTP-1 proteinsuch that the presence of MTP-1 protein or nucleic acid is detected inthe biological sample. A preferred agent for detecting MTP-1 mRNA orgenomic DNA is a labeled nucleic acid probe capable of hybridizing toMTP-1 mRNA or genomic DNA. The nucleic acid probe can be, for example,the MTP-1 nucleic acid set forth in SEQ ID NO:1 or 3, or a portionthereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or500 nucleotides in length and sufficient to specifically hybridize understringent conditions to MTP-1 mRNA or genomic DNA. Other suitable probesfor use in the diagnostic assays of the invention are described herein.

2. Regarding OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and HST-5

An exemplary method for detecting the presence or absence of OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5 polypeptides or nucleic acids in a biological sample involvesobtaining a biological sample from a test subject and contacting thebiological sample with a compound or an agent capable of detecting OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 polypeptides or nucleic acids (e.g., mRNA, or genomic DNA)that encodes OAT, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT,HST-4 and/or HST-5 polypeptides such that the presence of OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5 polypeptides or nucleic acids is detected in the biologicalsample. In another aspect, the present invention provides a method fordetecting the presence of OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 activity in a biological sampleby contacting the biological sample with an agent capable of detectingan indicator of OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 activity such that the presence of OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 activity is detected in the biological sample. A preferredagent for detecting OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 mRNA or genomic DNA is a labelednucleic acid probe capable of hybridizing to OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 mRNA orgenomic DNA. The nucleic acid probe can be, for example, the OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or theHST-5 nucleic acid set forth in SEQ ID NO:4, 6, 7, 9, 12, 14, 15, 17,19, 21, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 51, 53, 54, 56, 57, or59, or a portion thereof, such as an oligonucleotide of at least 15, 30,50, 100, 250 or 500 nucleotides in length and sufficient to specificallyhybridize under stringent conditions to OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 mRNA or genomicDNA. Other suitable probes for use in the diagnostic assays of theinvention are described herein.

3. Diagnostic Assays

A preferred agent for detecting MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 polypeptides is anantibody capable of binding to MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 polypeptides,preferably an antibody with a detectable label. Antibodies can bepolyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., Fab or F(ab′)2) can be used. The term “labeled”,with regard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with another reagentthat is directly labeled. Examples of indirect labeling includedetection of a primary antibody using a fluorescently labeled secondaryantibody and end-labeling of a DNA probe with biotin such that it can bedetected with fluorescently labeled streptavidin. The term “biologicalsample” is intended to include tissues, cells and biological fluidsisolated from a subject, as well as tissues, cells and fluids presentwithin a subject. That is, the detection method of the invention can beused to detect MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 mRNA, polypeptides, or genomicDNA in a biological sample in vitro as well as in vivo. For example, invitro techniques for detection of MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 mRNA includeNorthern hybridizations and in situ hybridizations. In vitro techniquesfor detection of MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 polypeptides include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. In vitro techniques fordetection of MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 genomic DNA include Southernhybridizations. Furthermore, in vivo techniques for detection of MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 polypeptides include introducing into a subject a labeledanti-MTP-1, anti-OAT, anti-HST-1, anti-TP-2, anti-PLTR-1, anti-TFM-2,anti-TFM-3, anti-67118, anti-67067, anti-62092, anti-HAAT, anti-HST-4and/or anti-HST-5 antibody. For example, the antibody can be labeledwith a radioactive marker whose presence and location in a subject canbe detected by standard imaging techniques.

The present invention also provides diagnostic assays for identifyingthe presence or absence of a genetic alteration characterized by atleast one of (i) aberrant modification or mutation of a gene encoding anOAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 polypeptide; (ii) aberrant expression of a gene encoding anOAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 polypeptide; (iii) mis-regulation of the gene; and (iv)aberrant post-translational modification of an OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 polypeptide,wherein a wild-type form of the gene encodes a polypeptide with an OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 an activity. “Misexpression or aberrant expression”, asused herein, refers to a non-wild type pattern of gene expression, atthe RNA or protein level. It includes, but is not limited to, expressionat non-wild type levels (e.g., over or under expression); a pattern ofexpression that differs from wild type in terms of the time or stage atwhich the gene is expressed (e.g., increased or decreased expression (ascompared with wild type) at a predetermined developmental period orstage); a pattern of expression that differs from wild type in terms ofdecreased expression (as compared with wild type) in a predeterminedcell type or tissue type; a pattern of expression that differs from wildtype in terms of the splicing size, amino acid sequence,post-transitional modification, or biological activity of the expressedpolypeptide; a pattern of expression that differs from wild type interms of the effect of an environmental stimulus or extracellularstimulus on expression of the gene (e.g., a pattern of increased ordecreased expression (as compared with wild type) in the presence of anincrease or decrease in the strength of the stimulus).

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a serum sample isolated byconventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5polypeptides, mRNA, or genomic DNA, such that the presence of MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 polypeptides, mRNA or genomic DNA is detected in thebiological sample, and comparing the presence of MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5 polypeptides, mRNA or genomic DNA in the control sample with thepresence of MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 polypeptides, mRNA or genomic DNA in thetest sample.

The invention also encompasses kits for detecting the presence of MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 in a biological sample. For example, the kit can comprise alabeled compound or agent capable of detecting MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5polypeptides or mRNA in a biological sample; means for determining theamount of MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 in the sample; and means for comparingthe amount of MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 in the sample with a standard.The compound or agent can be packaged in a suitable container. The kitcan further comprise instructions for using the kit to detect MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 polypeptides or nucleic acid.

4. Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant or unwanted MTP-1 expression or activity. Asused herein, the term “aberrant” includes an MTP-1 expression oractivity which deviates from the wild type MTP-1 expression or activity.Aberrant expression or activity includes increased or decreasedexpression or activity, as well as expression or activity which does notfollow the wild type developmental pattern of expression or thesubcellular pattern of expression. For example, aberrant MTP-1expression or activity is intended to include the cases in which amutation in the MTP-1 gene causes the MTP-1 gene to be under-expressedor over-expressed and situations in which such mutations result in anon-functional MTP-1 protein or a protein which does not function in awild-type fashion, e.g., a protein which does not interact with an MTP-1substrate, or one which interacts with a non-MTP-1 substrate. As usedherein, the term “unwanted” includes an unwanted phenomenon involved ina biological response such as inflammation and/or lipid metabolism. Forexample, the term unwanted includes an MTP-1 expression or activitywhich is undesirable in a subject.

The assays described herein, such as the preceding diagnostic assays orthe following assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with a misregulation in MTP-1protein activity or nucleic acid expression, such as a hematopoieticand/or immunological and/or lipid metabolism-related disorder, a CNSdisorder (e.g., a cognitive or neurodegenerative disorder), a cellularproliferation, growth, differentiation, or migration disorder, acardiovascular disorder, musculoskeletal disorder, an immune disorder,or a hormonal disorder. Alternatively, the prognostic assays can beutilized to identify a subject having or at risk for developing adisorder associated with a misregulation in MTP-1 protein activity ornucleic acid expression, such as a hematopoietic disorder, animmunological disorder, a lipid metabolism-related disorder, a CNSdisorder, a cellular proliferation, growth, differentiation, ormigration disorder, a musculoskeletal disorder, a cardiovasculardisorder, an immune disorder, or a hormonal disorder. Thus, the presentinvention provides a method for identifying a disease or disorderassociated with aberrant or unwanted MTP-1 expression or activity inwhich a test sample is obtained from a subject and MTP-1 protein ornucleic acid (e.g., mRNA or genomic DNA) is detected, wherein thepresence of MTP-1 protein or nucleic acid is diagnostic for a subjecthaving or at risk of developing a disease or disorder associated withaberrant or unwanted MTP-1 expression or activity. As used herein, a“test sample” refers to a biological sample obtained from a subject ofinterest. For example, a test sample can be a biological fluid (e.g.,cerebrospinal fluid or serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant or unwanted MTP-1 expression or activity. Forexample, such methods can be used to determine whether a subject can beeffectively treated with an agent for a hematopoietic disorder, animmunological disorder, a lipid metabolism-related disorder, a CNSdisorder, a muscular disorder, a cellular proliferation, growth,differentiation, or migration disorder, an immune disorder, or ahormonal disorder. Thus, the present invention provides methods fordetermining whether a subject can be effectively treated with an agentfor a disorder associated with aberrant or unwanted MTP-1 expression oractivity in which a test sample is obtained and MTP-1 protein or nucleicacid expression or activity is detected (e.g., wherein the abundance ofMTP-1 protein or nucleic acid expression or activity is diagnostic for asubject that can be administered the agent to treat a disorderassociated with aberrant or unwanted MTP-1 expression or activity).

The methods of the invention can also be used to detect geneticalterations in an MTP-1 gene, thereby determining if a subject with thealtered gene is at risk for a disorder characterized by misregulation inMTP-1 protein activity or nucleic acid expression, such as ahematopoietic disorder, an immunological disorder, a lipidmetabolism-related disorder, a CNS disorder, a musculoskeletal disorder,a cellular proliferation, growth, differentiation, or migrationdisorder, a cardiovascular disorder, an immune disorder, or a hormonaldisorder. In preferred embodiments, the methods include detecting, in asample of cells from the subject, the presence or absence of a geneticalteration characterized by at least one of an alteration affecting theintegrity of a gene encoding an MTP-1-protein, or the mis-expression ofthe MTP-1 gene. For example, such genetic alterations can be detected byascertaining the existence of at least one of 1) a deletion of one ormore nucleotides from an MTP-1 gene; 2) an addition of one or morenucleotides to an MTP-1 gene; 3) a substitution of one or morenucleotides of an MTP-1 gene, 4) a chromosomal rearrangement of an MTP-1gene; 5) an alteration in the level of a messenger RNA transcript of anMTP-1 gene, 6) aberrant modification of an MTP-1 gene, such as of themethylation pattern of the genomic DNA, 7) the presence of a non-wildtype splicing pattern of a messenger RNA transcript of an MTP-1 gene, 8)a non-wild type level of an MTP-1-protein, 9) allelic loss of an MTP-1gene, and 10) inappropriate post-translational modification of anMTP-1-protein. As described herein, there are a large number of assaysknown in the art which can be used for detecting alterations in an MTP-1gene. A preferred biological sample is a tissue or serum sample isolatedby conventional means from a subject.

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant or unwanted OAT expression or activity. As usedherein, the term “aberrant” includes an OAT expression or activity whichdeviates from the wild type OAT expression or activity. Aberrantexpression or activity includes increased or decreased expression oractivity, as well as expression or activity which does not follow thewild type developmental pattern of expression or the subcellular patternof expression. For example, aberrant OAT expression or activity isintended to include the cases in which a mutation in the OAT gene causesthe OAT gene to be under-expressed or over-expressed and situations inwhich such mutations result in a non-functional OAT protein or a proteinwhich does not function in a wild-type fashion, e.g., a protein whichdoes not interact with or transport an OAT substrate or target molecule,or one which interacts with a non-OAT substrate or target molecule. Asused herein, the term “unwanted” includes an unwanted phenomenoninvolved in a biological response such as the improper cellularlocalization of an OAT substrate or deregulated cell proliferation. Forexample, the term unwanted includes an OAT expression or activity whichis undesirable in a subject.

The assays described herein, such as the preceding diagnostic assays orthe following assays, can be utilized to identify a subject having or atrisk of developing an OAT associated disorder, e.g., a disorderassociated with a misregulation in OAT protein activity or nucleic acidexpression, such as a CNS disorder (e.g., a cognitive orneurodegenerative disorder), a cellular proliferation, growth,differentiation, or migration disorder, a cardiovascular disorder, amusculoskeletal disorder, an immune disorder, or a hormonal disorder.Alternatively, the prognostic assays can be utilized to identify asubject having or at risk for developing a disorder associated with amisregulation in OAT protein activity or nucleic acid expression, suchas a CNS disorder (e.g., a cognitive or neurodegenerative disorder), acellular proliferation, growth, differentiation, or migration disorder,a cardiovascular disorder, a musculoskeletal disorder, an immunedisorder, or a hormonal disorder. Thus, the present invention provides amethod for identifying a disease or disorder associated with aberrant orunwanted OAT expression or activity in which a test sample is obtainedfrom a subject and OAT protein or nucleic acid (e.g., mRNA or genomicDNA) is detected, wherein the presence of OAT protein or nucleic acid isdiagnostic for a subject having or at risk of developing a disease ordisorder associated with aberrant or unwanted OAT expression oractivity. As used herein, a “test sample” refers to a biological sampleobtained from a subject of interest. For example, a test sample can be abiological fluid (e.g., serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant or unwanted OAT expression or activity. Forexample, such methods can be used to determine whether a subject can beeffectively treated with an agent for a CNS disorder (e.g., a cognitiveor neurodegenerative disorder), a cellular proliferation, growth,differentiation, or migration disorder, a cardiovascular disorder, amusculoskeletal disorder, an immune disorder, or a hormonal disorder.Thus, the present invention provides methods for determining whether asubject can be effectively treated with an agent for a disorderassociated with aberrant or unwanted OAT expression or activity in whicha test sample is obtained and OAT protein or nucleic acid expression oractivity is detected (e.g., wherein the abundance of OAT protein ornucleic acid expression or activity is diagnostic for a subject that canbe administered the agent to treat a disorder associated with aberrantor unwanted OAT expression or activity).

The methods of the invention can also be used to detect geneticalterations in an OAT gene, thereby determining if a subject with thealtered gene is at risk for a disorder characterized by misregulation inOAT protein activity or nucleic acid expression, such as a CNS disorder(e.g., a cognitive or neurodegenerative disorder), a cellularproliferation, growth, differentiation, or migration disorder, acardiovascular disorder, a musculoskeletal disorder, an immune disorder,or a hormonal disorder. In preferred embodiments, the methods includedetecting, in a sample of cells from the subject, the presence orabsence of a genetic alteration characterized by at least one of analteration affecting the integrity of a gene encoding an OAT-protein, orthe mis-expression of the OAT gene. For example, such geneticalterations can be detected by ascertaining the existence of at leastone of 1) a deletion of one or more nucleotides from an OAT gene; 2) anaddition of one or more nucleotides to an OAT gene; 3) a substitution ofone or more nucleotides of an OAT gene, 4) a chromosomal rearrangementof an OAT gene; 5) an alteration in the level of a messenger RNAtranscript of an OAT gene, 6) aberrant modification of an OAT gene, suchas of the methylation pattern of the genomic DNA, 7) the presence of anon-wild type splicing pattern of a messenger RNA transcript of an OATgene, 8) a non-wild type level of an OAT-protein, 9) allelic loss of anOAT gene, and 10) inappropriate post-translational modification of anOAT-protein. As described herein, there are a large number of assaysknown in the art which can be used for detecting alterations in an OATgene. A preferred biological sample is a tissue or serum sample isolatedby conventional means from a subject.

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant or unwanted HST-1 expression or activity. Asused herein, the term “aberrant” includes an HST-1 expression oractivity which deviates from the wild type HST-1 expression or activity.Aberrant expression or activity includes increased or decreasedexpression or activity, as well as expression or activity which does notfollow the wild type developmental pattern of expression or thesubcellular pattern of expression. For example, aberrant HST-1expression, or activity is intended to include the cases in which amutation in the HST-1 gene causes the HST-1 gene to be under-expressedor over-expressed and situations in which such mutations result in anon-functional HST-1 polypeptide or a polypeptide which does notfunction in a wild-type fashion, e.g., a polypeptide which does notinteract with an HST-1 substrate, e.g., a sugar transporter subunit orligand, or one which interacts with a non-HST-1 substrate, e.g. anon-sugar transporter subunit or ligand. As used herein, the term“unwanted” includes an unwanted phenomenon involved in a biologicalresponse, such as cellular proliferation. For example, the term unwantedincludes an HST-1 expression or activity which is undesirable in asubject.

The assays described herein, such as the preceding diagnostic assays orthe following assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with a misregulation in HST-1polypeptide activity or nucleic acid expression, such as a sugartransporter disorder. Alternatively, the prognostic assays can beutilized to identify a subject having or at risk for developing adisorder associated with a misregulation in HST-1 polypeptide activityor nucleic acid expression, such as a sugar transporter disorder. Thus,the present invention provides a method for identifying a disease ordisorder associated with aberrant or unwanted HST-1 expression oractivity in which a test sample is obtained from a subject and HST-1polypeptide or nucleic acid (e.g., mRNA or genomic DNA) is detected,wherein the presence of HST-1 polypeptide or nucleic acid is diagnosticfor a subject having or at risk of developing a disease or disorderassociated with aberrant or unwanted HST-1 expression or activity. Asused herein, a “test sample” refers to a biological sample obtained froma subject of interest. For example, a test sample can be a biologicalfluid (e.g., serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant or unwanted HST-1 expression or activity. Forexample, such methods can be used to determine whether a subject can beeffectively treated with an agent for a sugar transporter disorder.Thus, the present invention provides methods for determining whether asubject can be effectively treated with an agent for a disorderassociated with aberrant or unwanted HST-1 expression or activity inwhich a test sample is obtained and HST-1 polypeptide or nucleic acidexpression or activity is detected (e.g., wherein the abundance of HST-1polypeptide or nucleic acid expression or activity is diagnostic for asubject that can be administered the agent to treat a disorderassociated with aberrant or unwanted HST-1 expression or activity).

The methods of the invention can also be used to detect geneticalterations in an HST-1 gene, thereby determining if a subject with thealtered gene is at risk for a disorder characterized by misregulation inHST-1 polypeptide activity or nucleic acid expression, such as a sugartransporter disorder, a sugar homeostasis disorder, or a disorder ofcellular growth, differentiation, or migration. In preferredembodiments, the methods include detecting, in a sample of cells fromthe subject, the presence or absence of a genetic alterationcharacterized by at least one of an alteration affecting the integrityof a gene encoding an HST-1-polypeptide, or the mis-expression of theHST-1 gene. For example, such genetic alterations can be detected byascertaining the existence of at least one of 1) a deletion of one ormore nucleotides from an HST-1 gene; 2) an addition of one or morenucleotides to an HST-1 gene; 3) a substitution of one or morenucleotides of an HST-1 gene, 4) a chromosomal rearrangement of an HST-1gene; 5) an alteration in the level of a messenger RNA transcript of anHST-1 gene, 6) aberrant modification of an HST-1 gene, such as of themethylation pattern of the genomic DNA, 7) the presence of a non-wildtype splicing pattern of a messenger RNA transcript of an HST-1 gene, 8)a non-wild type level of an HST-1-polypeptide, 9) allelic loss of anHST-1 gene, and 10) inappropriate post-translational modification of anHST-1-polypeptide. As described herein, there are a large number ofassays known in the art which can be used for detecting alterations inan HST-1 gene. A preferred biological sample is a tissue or serum sampleisolated by conventional means from a subject.

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant or unwanted TP-2 expression or activity. Asused herein, the term “aberrant” includes a TP-2 expression or activitywhich deviates from the wild type TP-2 expression or activity. Aberrantexpression or activity includes increased or decreased expression oractivity, as well as expression or activity which does not follow thewild type developmental pattern of expression or the subcellular patternof expression. For example, aberrant TP-2 expression or activity isintended to include the cases in which a mutation in the TP-2 genecauses the TP-2 gene to be under-expressed or over-expressed andsituations in which such mutations result in a non-functional TP-2polypeptide or a polypeptide which does not function in a wild-typefashion, e.g., a polypeptide which does not interact with a TP-2substrate, e.g., a transporter subunit or ligand, or one which interactswith a non-TP-2 substrate, e.g. a non-transporter subunit or ligand. Asused herein, the term “unwanted” includes an unwanted phenomenoninvolved in a biological response, such as cellular proliferation. Forexample, the term unwanted includes a TP-2 expression or activity whichis undesirable in a subject.

The assays described herein, such as the preceding diagnostic assays orthe following assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with a misregulation in TP-2polypeptide activity or nucleic acid expression, such as atransporter-associated disorder. Alternatively, the prognostic assayscan be utilized to identify a subject having or at risk for developing adisorder associated with a misregulation in TP-2 polypeptide activity ornucleic acid expression, such as a transporter-associated disorder.Thus, the present invention provides a method for identifying a diseaseor disorder associated with aberrant or unwanted TP-2 expression oractivity in which a test sample is obtained from a subject and TP-2polypeptide or nucleic acid (e.g., mRNA or genomic DNA) is detected,wherein the presence of TP-2 polypeptide or nucleic acid is diagnosticfor a subject having or at risk of developing a disease or disorderassociated with aberrant or unwanted TP-2 expression or activity. Asused herein, a “test sample” refers to a biological sample obtained froma subject of interest. For example, a test sample can be a biologicalfluid (e.g., serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant or unwanted TP-2 expression or activity. Forexample, such methods can be used to determine whether a subject can beeffectively treated with an agent for a transporter-associated disorder.Thus, the present invention provides methods for determining whether asubject can be effectively treated with an agent for a disorderassociated with aberrant or unwanted TP-2 expression or activity inwhich a test sample is obtained and TP-2 polypeptide or nucleic acidexpression or activity is detected (e.g., wherein the abundance of TP-2polypeptide or nucleic acid expression or activity is diagnostic for asubject that can be administered the agent to treat a disorderassociated with aberrant or unwanted TP-2 expression or activity).

The methods of the invention can also be used to detect geneticalterations in a TP-2 gene, thereby determining if a subject with thealtered gene is at risk for a disorder characterized by misregulation inTP-2 polypeptide activity or nucleic acid expression, such as atransporter-associated disorder. In preferred embodiments, the methodsinclude detecting, in a sample of cells from the subject, the presenceor absence of a genetic alteration characterized by at least one of analteration affecting the integrity of a gene encoding a TP-2-polypeptide, or the mis-expression of the TP-2 gene. For example, suchgenetic alterations can be detected by ascertaining the existence of atleast one of 1) a deletion of one or more nucleotides from a TP-2 gene;2) an addition of one or more nucleotides to a TP-2 gene; 3) asubstitution of one or more nucleotides of a TP-2 gene, 4) a chromosomalrearrangement of a TP-2 gene; 5) an alteration in the level of amessenger RNA transcript of a TP-2 gene, 6) aberrant modification of aTP-2 gene, such as of the methylation pattern of the genomic DNA, 7) thepresence of a non-wild type splicing pattern of a messenger RNAtranscript of a TP-2 gene, 8) a non-wild type level of aTP-2-polypeptide, 9) allelic loss of a TP-2 gene, and 10) inappropriatepost-translational modification of a TP-2-polypeptide. As describedherein, there are a large number of assays known in the art which can beused for detecting alterations in a TP-2 gene. A preferred biologicalsample is a tissue or serum sample isolated by conventional means from asubject.

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant or unwanted PLTR-1 expression or activity(e.g., a cardiovascular disorder). As used herein, the term “aberrant”includes a PLTR-1 expression or activity which deviates from the wildtype PLTR-1 expression or activity. Aberrant expression or activityincludes increased or decreased expression or activity, as well asexpression or activity which does not follow the wild type developmentalpattern of expression or the subcellular pattern of expression. Forexample, aberrant PLTR-1 expression or activity is intended to includethe cases in which a mutation in the PLTR-1 gene causes the PLTR-1 geneto be under-expressed or over-expressed and situations in which suchmutations result in a non-functional PLTR-1 protein or a protein whichdoes not function in a wild-type fashion, e.g., a protein which does notinteract with or transport a PLTR-1 substrate, or one which interactswith or transports a non-PLTR-1 substrate. As used herein, the term“unwanted” includes an unwanted phenomenon involved in a biologicalresponse such as deregulated cell proliferation. For example, the termunwanted includes a PLTR-1 expression or activity which is undesirablein a subject.

The assays described herein, such as the preceding diagnostic assays orthe following assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with a misregulation in PLTR-1protein activity or nucleic acid expression, such as a cardiovasculardisorder or a cell growth, proliferation and/or differentiationdisorder. Alternatively, the prognostic assays can be utilized toidentify a subject having or at risk for developing a disorderassociated with a misregulation in PLTR-1 protein activity or nucleicacid expression, such as a cardiovascular disorder or a cell growth,proliferation and/or differentiation disorder. Thus, the presentinvention provides a method for identifying a disease or disorderassociated with aberrant or unwanted PLTR-1 expression or activity inwhich a test sample is obtained from a subject and PLTR-1 protein ornucleic acid (e.g., mRNA or genomic DNA) is detected, wherein thepresence of PLTR-1 protein or nucleic acid is diagnostic for a subjecthaving or at risk of developing a disease or disorder associated withaberrant or unwanted PLTR-1 expression or activity. As used herein, a“test sample” refers to a biological sample obtained from a subject ofinterest. For example, a test sample can be a biological fluid (e.g.,serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant or unwanted PLTR-1 expression or activity(e.g., a cardiovascular disorder). For example, such methods can be usedto determine whether a subject can be effectively treated with an agentfor a cardiovascular disorder, a drug or toxin sensitivity disorder, ora cell proliferation and/or differentiation disorder. Thus, the presentinvention provides methods for determining whether a subject can beeffectively treated with an agent for a disorder associated withaberrant or unwanted PLTR-1 expression or activity in which a testsample is obtained and PLTR-1 protein or nucleic acid expression oractivity is detected (e.g., wherein the abundance of PLTR-1 protein ornucleic acid expression or activity is diagnostic for a subject that canbe administered the agent to treat a disorder associated with aberrantor unwanted PLTR-1 expression or activity).

The methods of the invention can also be used to detect geneticalterations in a PLTR-1 gene, thereby determining if a subject with thealtered gene is at risk for a disorder characterized by misregulation inPLTR-1 protein activity or nucleic acid expression, such as acardiovascular disorder or a cell growth, proliferation and/ordifferentiation disorder. In preferred embodiments, the methods includedetecting, in a sample of cells from the subject, the presence orabsence of a genetic alteration characterized by at least one of analteration affecting the integrity of a gene encoding a PLTR-1-protein,or the mis-expression of the PLTR-1 gene. For example, such geneticalterations can be detected by ascertaining the existence of at leastone of 1) a deletion of one or more nucleotides from a PLTR-1 gene; 2)an addition of one or more nucleotides to a PLTR-1 gene; 3) asubstitution of one or more nucleotides of a PLTR-1 gene, 4) achromosomal rearrangement of a PLTR-1 gene; 5) an alteration in thelevel of a messenger RNA transcript of a PLTR-1 gene, 6) aberrantmodification of a PLTR-1 gene, such as of the methylation pattern of thegenomic DNA, 7) the presence of a non-wild type splicing pattern of amessenger RNA transcript of a PLTR-1 gene, 8) a non-wild type level of aPLTR-1-protein, 9) allelic loss of a PLTR-1 gene, and 10) inappropriatepost-translational modification of a PLTR-1-protein. As describedherein, there are a large number of assays known in the art which can beused for detecting alterations in a PLTR-1 gene. A preferred biologicalsample is a tissue or serum sample isolated by conventional means from asubject.

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant or unwanted TFM-2 and/or TFM-3 expression oractivity. As used herein, the term “aberrant” includes a TFM-2 and/orTFM-3 expression or activity which deviates from the wild type TFM-2and/or TFM-3 expression or activity. Aberrant expression or activityincludes increased or decreased expression or activity, as well asexpression or activity which does not follow the wild type developmentalpattern of expression or the subcellular pattern of expression. Forexample, aberrant TFM-2 and/or TFM-3 expression or activity is intendedto include the cases in which a mutation in the TFM-2 and/or TFM-3 genecauses the TFM-2 and/or TFM-3 gene to be under-expressed orover-expressed and situations in which such mutations result in anon-functional TFM-2 and/or TFM-3 polypeptide or a polypeptide whichdoes not function in a wild-type fashion, e.g., a polypeptide which doesnot interact with a TFM-2 and/or TFM-3 substrate, e.g., a transportersubunit or ligand, or one which interacts with a non-TFM-2 and/or TFM-3substrate, e.g. a non-transporter subunit or ligand. As used herein, theterm “unwanted” includes an unwanted phenomenon involved in a biologicalresponse, such as cellular proliferation. For example, the term unwantedincludes a TFM-2 and/or TFM-3 expression or activity which isundesirable in a subject.

The assays described herein, such as the preceding diagnostic assays orthe following assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with a misregulation in TFM-2and/or TFM-3 polypeptide activity or nucleic acid expression, such as atransporter-associated disorder. Alternatively, the prognostic assayscan be utilized to identify a subject having or at risk for developing adisorder associated with a misregulation in TFM-2 and/or TFM-3polypeptide activity or nucleic acid expression, such as atransporter-associated disorder. Thus, the present invention provides amethod for identifying a disease or disorder associated with aberrant orunwanted TFM-2 and/or TFM-3 expression or activity in which a testsample is obtained from a subject and TFM-2 and/or TFM-3 polypeptide ornucleic acid (e.g., mRNA or genomic DNA) is detected, wherein thepresence of TFM-2 and/or TFM-3 polypeptide or nucleic acid is diagnosticfor a subject having or at risk of developing a disease or disorderassociated with aberrant or unwanted TFM-2 and/or TFM-3 expression oractivity. As used herein, a “test sample” refers to a biological sampleobtained from a subject of interest. For example, a test sample can be abiological fluid (e.g., serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant or unwanted TFM-2 and/or TFM-3 expression oractivity. For example, such methods can be used to determine whether asubject can be effectively treated with an agent for atransporter-associated disorder. Thus, the present invention providesmethods for determining whether a subject can be effectively treatedwith an agent for a disorder associated with aberrant or unwanted TFM-2and/or TFM-3 expression or activity in which a test sample is obtainedand TFM-2 and/or TFM-3 polypeptide or nucleic acid expression oractivity is detected (e.g., wherein the abundance of TFM-2 and/or TFM-3polypeptide or nucleic acid expression or activity is diagnostic for asubject that can be administered the agent to treat a disorderassociated with aberrant or unwanted TFM-2 and/or TFM-3 expression oractivity).

The methods of the invention can also be used to detect geneticalterations in a TFM-2 and/or TFM-3 gene, thereby determining if asubject with the altered gene is at risk for a disorder characterized bymisregulation in TFM-2 and/or TFM-3 polypeptide activity or nucleic acidexpression, such as a transporter-associated disorder. In preferredembodiments, the methods include detecting, in a sample of cells fromthe subject, the presence or absence of a genetic alterationcharacterized by at least one of an alteration affecting the integrityof a gene encoding a TFM-2 and/or TFM-3-polypeptide, or themis-expression of the TFM-2 and/or TFM-3 gene. For example, such geneticalterations can be detected by ascertaining the existence of at leastone of 1) a deletion of one or more nucleotides from a TFM-2 and/orTFM-3 gene; 2) an addition of one or more nucleotides to a TFM-2 and/orTFM-3 gene; 3) a substitution of one or more nucleotides of a TFM-2and/or TFM-3 gene, 4) a chromosomal rearrangement of a TFM-2 and/orTFM-3 gene; 5) an alteration in the level of a messenger RNA transcriptof a TFM-2 and/or TFM-3 gene, 6) aberrant modification of a TFM-2 and/orTFM-3 gene, such as of the methylation pattern of the genomic DNA, 7)the presence of a non-wild type splicing pattern of a messenger RNAtranscript of a TFM-2 and/or TFM-3 gene, 8) a non-wild type level of aTFM-2 and/or TFM-3-polypeptide, 9) allelic loss of a TFM-2 and/or TFM-3gene, and 10) inappropriate post-translational modification of a TFM-2and/or TFM-3-polypeptide. As described herein, there are a large numberof assays known in the art which can be used for detecting alterationsin a TFM-2 and/or TFM-3 gene. A preferred biological sample is a tissueor serum sample isolated by conventional means from a subject.

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant or unwanted 67118, 67067, and/or 62092expression or activity. As used herein, the term “aberrant” includes a67118, 67067, and/or 62092 expression or activity which deviates fromthe wild type 67118, 67067, and/or 62092 expression or activity.Aberrant expression or activity includes increased or decreasedexpression or activity, as well as expression or activity which does notfollow the wild type developmental pattern of expression or thesubcellular pattern of expression. For example, aberrant 67118, 67067,and/or 62092 expression or activity is intended to include the cases inwhich a mutation in the 67118, 67067, and/or 62092 gene causes the67118, 67067, and/or 62092 gene to be under-expressed or over-expressedand situations in which such mutations result in a non-functional 67118,67067, and/or 62092 polypeptide or a polypeptide which does not functionin a wild-type fashion, e.g., a protein which does not interact with ortransport a 67118, 67067, and/or 62092 substrate, or one which interactswith or transports a non-67118, 67067, and/or 62092 substrate. As usedherein, the term “unwanted” includes an unwanted phenomenon involved ina biological response such as deregulated cell proliferation. Forexample, the term unwanted includes a 67118, 67067, and/or 62092expression or activity which is undesirable in a subject.

The assays described herein, such as the preceding diagnostic assays orthe following assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with a misregulation in 67118,67067, and/or 62092 polypeptide activity or nucleic acid expression,such as a as a cell growth, proliferation and/or differentiationdisorder, e.g., cancer, including, but not limited to colon cancer orlung cancer. Alternatively, the prognostic assays can be utilized toidentify a subject having or at risk for developing a disorderassociated with a misregulation in 67118, 67067, and/or 62092polypeptide activity or nucleic acid expression, such as a cell growth,proliferation and/or differentiation disorder. Thus, the presentinvention provides a method for identifying a disease or disorderassociated with aberrant or unwanted 67118, 67067, and/or 62092expression or activity in which a test sample is obtained from a subjectand 67118, 67067, and/or 62092 polypeptide or nucleic acid (e.g., mRNAor genomic DNA) is detected, wherein the presence of 67118, 67067,and/or 62092 polypeptide or nucleic acid is diagnostic for a subjecthaving or at risk of developing a disease or disorder associated withaberrant or unwanted 67118, 67067, and/or 62092 expression or activity.As used herein, a “test sample” refers to a biological sample obtainedfrom a subject of interest. For example, a test sample can be abiological fluid (e.g., serum), cell sample, or tissue, e.g., a colontumor sample or a lung tumor sample.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant or unwanted 67118, 67067, and/or 62092expression or activity. For example, such methods can be used todetermine whether a subject can be effectively treated with an agent fora transporter-associated disorder. Thus, the present invention providesmethods for determining whether a subject can be effectively treatedwith an agent for a disorder associated with aberrant or unwanted 67118,67067, and/or 62092 expression or activity in which a test sample isobtained and 67118, 67067, and/or 62092 polypeptide or nucleic acidexpression or activity is detected (e.g., wherein the abundance of67118, 67067, and/or 62092 polypeptide or nucleic acid expression oractivity is diagnostic for a subject that can be administered the agentto treat a disorder associated with aberrant or unwanted 67118, 67067,and/or 62092 expression or activity).

The methods of the invention can also be used to detect geneticalterations in a 67118, 67067, and/or 62092 gene, thereby determining ifa subject with the altered gene is at risk for a disorder characterizedby misregulation in 67118, 67067, and/or 62092 polypeptide activity ornucleic acid expression, such as a cell growth, proliferation and/ordifferentiation disorder. In preferred embodiments, the methods includedetecting, in a sample of cells from the subject, the presence orabsence of a genetic alteration characterized by at least one of analteration affecting the integrity of a gene encoding a 67118, 67067,and/or 62092-polypeptide, or the mis-expression of the 67118, 67067,and/or 62092 gene. For example, such genetic alterations can be detectedby ascertaining the existence of at least one of 1) a deletion of one ormore nucleotides from a 67118, 67067, and/or 62092 gene; 2) an additionof one or more nucleotides to a 67118, 67067, and/or 62092 gene; 3) asubstitution of one or more nucleotides of a 67118, 67067, and/or 62092gene, 4) a chromosomal rearrangement of a 67118, 67067, and/or 62092gene; 5) an alteration in the level of a messenger RNA transcript of a67118, 67067, and/or 62092 gene, 6) aberrant modification of a 67118,67067, and/or 62092 gene, such as of the methylation pattern of thegenomic DNA, 7) the presence of a non-wild type splicing pattern of amessenger RNA transcript of a 67118, 67067, and/or 62092 gene, 8) anon-wild type level of a 67118, 67067, and/or 62092-polypeptide, 9)allelic loss of a 67118, 67067, and/or 62092 gene, and 10) inappropriatepost-translational modification of a 67118, 67067, and/or62092-polypeptide. As described herein, there are a large number ofassays known in the art which can be used for detecting alterations in a67118, 67067, and/or 62092 gene. A preferred biological sample is atissue or serum sample isolated by conventional means from a subject.

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant or unwanted HAAT expression or activity. Asused herein, the term “aberrant” includes a HAAT expression or activitywhich deviates from the wild type HAAT expression or activity. Aberrantexpression or activity includes increased or decreased expression oractivity, as well as expression or activity which does not follow thewild type developmental pattern of expression or the subcellular patternof expression. For example, aberrant HAAT expression or activity isintended to include the cases in which a mutation in the HAAT genecauses the HAAT gene to be under-expressed or over-expressed andsituations in which such mutations result in a non-functional HAATprotein or a protein which does not function in a wild-type fashion,e.g., a protein which does not interact with or transport a HAATsubstrate, or one which interacts with or transports a non-HAATsubstrate.

The assays described herein, such as the preceding diagnostic assays orthe following assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with a misregulation in HAATprotein activity or nucleic acid expression, such as tumorigenesisand/or nerve transmission. Alternatively, the prognostic assays can beutilized to identify a subject having or at risk for developing adisorder associated with a misregulation in HAAT protein activity ornucleic acid expression, such as a tumorigenesis and/or nervetransmission disorder. Thus, the present invention provides a method foridentifying a disease or disorder associated with aberrant or unwantedHAAT expression or activity in which a test sample is obtained from asubject and HAAT protein or nucleic acid (e.g., mRNA or genomic DNA) isdetected, wherein the presence of HAAT protein or nucleic acid isdiagnostic for a subject having or at risk of developing a disease ordisorder associated with aberrant or unwanted HAAT expression oractivity. As used herein, a “test sample” refers to a biological sampleobtained from a subject of interest. For example, a test sample can be abiological fluid (e.g., serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant or unwanted HAAT expression or activity. Forexample, such methods can be used to determine whether a subject can beeffectively treated with an agent for a drug or toxin sensitivitydisorder or a tumorigenesis and/or nerve transmission disorder. Thus,the present invention provides methods for determining whether a subjectcan be effectively treated with an agent for a disorder associated withaberrant or unwanted HAAT expression or activity in which a test sampleis obtained and HAAT protein or nucleic acid expression or activity isdetected (e.g., wherein the abundance of HAAT protein or nucleic acidexpression or activity is diagnostic for a subject that can beadministered the agent to treat a disorder associated with aberrant orunwanted HAAT expression or activity).

The methods of the invention can also be used to detect geneticalterations in a HAAT gene, thereby determining if a subject with thealtered gene is at risk for a disorder characterized by misregulation inHAAT protein activity or nucleic acid expression, such as atumorigenesis and/or nerve transmission disorder. In preferredembodiments, the methods include detecting, in a sample of cells fromthe subject, the presence or absence of a genetic alterationcharacterized by at least one of an alteration affecting the integrityof a gene encoding a HAAT-protein, or the mis-expression of the HAATgene. For example, such genetic alterations can be detected byascertaining the existence of at least one of 1) a deletion of one ormore nucleotides from a HAAT gene; 2) an addition of one or morenucleotides to a HAAT gene; 3) a substitution of one or more nucleotidesof a HAAT gene, 4) a chromosomal rearrangement of a HAAT gene; 5) analteration in the level of a messenger RNA transcript of a HAAT gene, 6)aberrant modification of a HAAT gene, such as of the methylation patternof the genomic DNA, 7) the presence of a non-wild type splicing patternof a messenger RNA transcript of a HAAT gene, 8) a non-wild type levelof a HAAT-protein, 9) allelic loss of a HAAT gene, and 10) inappropriatepost-translational modification of a HAAT-protein. As described herein,there are a large number of assays known in the art which can be usedfor detecting alterations in a HAAT gene. A preferred biological sampleis a tissue or serum sample isolated by conventional means from asubject.

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant or unwanted HST-4 and/or HST-5 expression oractivity. As used herein, the term “aberrant” includes an HST-4 and/oran HST-5 expression or activity which deviates from the wild type HST-4and/or HST-5 expression or activity. Aberrant expression or activityincludes increased or decreased expression or activity, as well asexpression or activity which does not follow the wild type developmentalpattern of expression or the subcellular pattern of expression. Forexample, aberrant HST-4 and/or HST-5 expression or activity is intendedto include the cases in which a mutation in the HST-4 and/or the HST-5gene causes the HST-4 and/or the HST-5 gene to be under-expressed orover-expressed and situations in which such mutations result in anon-functional HST-4 and/or HST-5 polypeptides or polypeptides which donot function in a wild-type fashion, e.g., polypeptides which do notinteract with an HST-4 and/or an HST-5 substrate, e.g., a sugartransporter subunit or ligand, or one which interacts with a non-HST-4and/or a non-HST-5 substrate, e.g. a non-sugar transporter subunit orligand. As used herein, the term “unwanted” includes an unwantedphenomenon involved in a biological response, such as cellularproliferation. For example, the term unwanted includes an HST-4 and/oran HST-5 expression or activity which is undesirable in a subject.

The assays described herein, such as the preceding diagnostic assays orthe following assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with a misregulation in HST-4and/or HST-5 polypeptide activity or nucleic acid expression, such as asugar transporter disorder. Alternatively, the prognostic assays can beutilized to identify a subject having or at risk for developing adisorder associated with a misregulation in HST-4 and/or HST-5polypeptide activity or nucleic acid expression, such as a sugartransporter disorder. Thus, the present invention provides a method foridentifying a disease or disorder associated with aberrant or unwantedHST-4 and/or HST-5 expression or activity in which a test sample isobtained from a subject and HST-4 and/or HST-5 polypeptides or nucleicacids (e.g., mRNA or genomic DNA) are detected, wherein the presence ofHST-4 and/or HST-5 polypeptides or nucleic acids are diagnostic for asubject having or at risk of developing a disease or disorder associatedwith aberrant or unwanted HST-4 and/or HST-5 expression or activity. Asused herein, a “test sample” refers to a biological sample obtained froma subject of interest. For example, a test sample can be a biologicalfluid (e.g., serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant or unwanted HST-4 and/or HST-5 expression oractivity. For example, such methods can be used to determine whether asubject can be effectively treated with an agent for a sugar transporterdisorder. Thus, the present invention provides methods for determiningwhether a subject can be effectively treated with an agent for adisorder associated with aberrant or unwanted HST-4 and/or HST-5expression or activity in which a test sample is obtained and HST-4and/or HST-5 polypeptide or nucleic acid expression or activity isdetected (e.g., wherein the abundance of HST-4 and/or HST-5 polypeptideor nucleic acid expression or activity is diagnostic for a subject thatcan be administered the agent to treat a disorder associated withaberrant or unwanted HST-4 and/or HST-5 expression or activity).

The methods of the invention can also be used to detect geneticalterations in an HST-4 and/or an HST-5 gene, thereby determining if asubject with the altered gene is at risk for a disorder characterized bymisregulation in HST-4 and/or HST-5 polypeptide activity or nucleic acidexpression, such as a sugar transporter disorder, a sugar homeostasisdisorder, or a disorder of cellular growth, differentiation, ormigration. In preferred embodiments, the methods include detecting, in asample of cells from the subject, the presence or absence of a geneticalteration characterized by at least one of an alteration affecting theintegrity of a gene encoding an HST-4-polypeptide and/or anHST-5-polypeptide, or the mis-expression of the HST-4 and/or the HST-5gene. For example, such genetic alterations can be detected byascertaining the existence of at least one of 1) a deletion of one ormore nucleotides from an HST-4 and/or an HST-5 gene; 2) an addition ofone or more nucleotides to an HST-4 and/or an HST-5 gene; 3) asubstitution of one or more nucleotides of an HST-4 and/or an HST-5gene, 4) a chromosomal rearrangement of an HST-4 and/or an HST-5 gene;5) an alteration in the level of a messenger RNA transcript of an HST-4and/or an HST-5 gene, 6) aberrant modification of an HST-4 and/or anHST-5 gene, such as of the methylation pattern of the genomic DNA, 7)the presence of a non-wild type splicing pattern of a messenger RNAtranscript of an HST-4 and/or an HST-5 gene, 8) a non-wild type level ofan HST-4-polypeptide and/or an HST-5-polypeptide, 9) allelic loss of anHST-4 and/or an HST-5 gene, and 10) inappropriate post-translationalmodification of an HST-4-polypeptide and/or an HST-5-polypeptide. Asdescribed herein, there are a large number of assays known in the artwhich can be used for detecting alterations in an HST-4 and/or an HST-5gene. A preferred biological sample is a tissue or serum sample isolatedby conventional means from a subject.

In certain embodiments, detection of the alteration involves the use ofa probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4-geneand/or the HST-5-gene (see Abravaya et al. (1995) Nucleic Acids Res.23:675-682). This method can include the steps of collecting a sample ofcells from a subject, isolating nucleic acid (e.g., genomic, mRNA orboth) from the cells of the sample, contacting the nucleic acid samplewith one or more primers which specifically hybridize to an MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or an HST-5 gene under conditions such that hybridization andamplification of the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4-gene and/or the HST-5-gene (if present)occurs, and detecting the presence or absence of an amplificationproduct, or detecting the size of the amplification product andcomparing the length to a control sample. It is anticipated that PCRand/or LCR may be desirable to use as a preliminary amplification stepin conjunction with any of the techniques used for detecting mutationsdescribed herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.,(1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al. (1988) Bio-Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in an MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or an HST-5gene from a sample cell can be identified by alterations in restrictionenzyme cleavage patterns. For example, sample and control DNA isisolated, amplified (optionally), digested with one or more restrictionendonucleases, and fragment length sizes are determined by gelelectrophoresis and compared. Differences in fragment length sizesbetween sample and control DNA indicates mutations in the sample DNA.Moreover, the use of sequence specific ribozymes (see, for example, U.S.Pat. No. 5,498,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 canbe identified by hybridizing a sample and control nucleic acids, e.g.,DNA or RNA, to high density arrays containing hundreds or thousands ofoligonucleotides probes (Cronin, M. T. et al. (1996) Human Mutation 7:244-255; Kozal, M. J. et al. (1996) Nature Medicine 2: 753-759). Forexample, genetic mutations in MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 can be identifiedin two dimensional arrays containing light-generated DNA probes asdescribed in Cronin, M. T. et al. supra. Briefly, a first hybridizationarray of probes can be used to scan through long stretches of DNA in asample and control to identify base changes between the sequences bymaking linear arrays of sequential overlapping probes. This step allowsthe identification of point mutations. This step is followed by a secondhybridization array that allows the characterization of specificmutations by using smaller, specialized probe arrays complementary toall variants or mutations detected. Each mutation array is composed ofparallel probe sets, one complementary to the wild-type gene and theother complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or theHST-5 gene and detect mutations by comparing the sequence of the sampleMTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5 with the corresponding wild-type (control)sequence. Examples of sequencing reactions include those based ontechniques developed by Maxam and Gilbert ((1977) Proc. Natl. Acad. Sci.USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It isalso contemplated that any of a variety of automated sequencingprocedures can be utilized when performing the diagnostic assays ((1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al. (1996)Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem.Biotechnol. 38:147-159).

Other methods for detecting mutations in the MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or the HST-5gene include methods in which protection from cleavage agents is used todetect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers etal. (1985) Science 230:1242). In general, the art technique of “mismatchcleavage” starts by providing heteroduplexes of formed by hybridizing(labeled) RNA or DNA containing the wild-type HST-4 and/or HST-5sequence with potentially mutant RNA or DNA obtained from a tissuesample. The double-stranded duplexes are treated with an agent whichcleaves single-stranded regions of the duplex such as which will existdue to basepair mismatches between the control and sample strands. Forinstance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybridstreated with S1 nuclease to enzymatically digesting the mismatchedregions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can betreated with hydroxylamine or osmium tetroxide and with piperidine inorder to digest mismatched regions. After digestion of the mismatchedregions, the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, for example,Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al.(1992) Methods Enzymol. 217:286-295. In a preferred embodiment, thecontrol DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5cDNAs obtained from samples of cells. For example, the mutY enzyme of E.coli cleaves A at G/A mismatches and the thymidine DNA glycosylase fromHeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis15:1657-1662). According to an exemplary embodiment, a probe based on anHST-4 and/or an HST-5 sequence, e.g., a wild-type MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5 sequence, is hybridized to a cDNA or other DNA product from a testcell(s). The duplex is treated with a DNA mismatch repair enzyme, andthe cleavage products, if any, can be detected from electrophoresisprotocols or the like. See, for example, U.S. Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 genes. For example,single strand conformation polymorphism (SSCP) may be used to detectdifferences in electrophoretic mobility between mutant and wild typenucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766,see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992)Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments ofsample and control MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 nucleic acids will be denaturedand allowed to renature. The secondary structure of single-strandednucleic acids varies according to sequence, the resulting alteration inelectrophoretic mobility enables the detection of even a single basechange. The DNA fragments may be labeled or detected with labeledprobes. The sensitivity of the assay may be enhanced by using RNA(rather than DNA), in which the secondary structure is more sensitive toa change in sequence. In a preferred embodiment, the subject methodutilizes heteroduplex analysis to separate double stranded heteroduplexmolecules on the basis of changes in electrophoretic mobility (Keen etal. (1991) Trends Genet 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci.USA 86:6230). Such allele specific oligonucleotides are hybridized toPCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving an MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5 gene.

Furthermore, any cell type or tissue in which MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 isexpressed may be utilized in the prognostic assays described herein.

5. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of an MTP-1 protein (e.g., the maintenance of cellularhomeostasis) can be applied not only in basic drug screening, but alsoin clinical trials. For example, the effectiveness of an agentdetermined by a screening assay as described herein to increase MTP-1gene expression, protein levels, or upregulate MTP-1 activity, can bemonitored in clinical trials of subjects exhibiting decreased MTP-1 geneexpression, protein levels, or downregulated MTP-1 activity.Alternatively, the effectiveness of an agent determined by a screeningassay to decrease MTP-1 gene expression, protein levels, or downregulateMTP-1 activity, can be monitored in clinical trials of subjectsexhibiting increased MTP-1 gene expression, protein levels, orupregulated MTP-1 activity. In such clinical trials, the expression oractivity of an MTP-1 gene, and preferably, other genes that have beenimplicated in, for example, an MTP-1-associated disorder can be used asa “read out” or markers of the phenotype of a particular cell.

For example, and not by way of limitation, genes, including MTP-1, thatare modulated in cells by treatment with an agent (e.g., compound, drugor small molecule) which modulates MTP-1 activity (e.g., identified in ascreening assay as described herein) can be identified. Thus, to studythe effect of agents on MTP-1-associated disorders (e.g., disorderscharacterized by deregulated hematopoiesis and/or inflammation and/orlipid metabolism), for example, in a clinical trial, cells can beisolated and RNA prepared and analyzed for the levels of expression ofMTP-1 and other genes implicated in the MTP-1-associated disorder,respectively. The levels of gene expression (e.g., a gene expressionpattern) can be quantified by northern blot analysis or RT-PCR, asdescribed herein, or alternatively by measuring the amount of proteinproduced, by one of the methods as described herein, or by measuring thelevels of activity of MTP-1 or other genes. In this way, the geneexpression pattern can serve as a marker, indicative of thephysiological response of the cells to the agent. Accordingly, thisresponse state may be determined before, and at various points duringtreatment of the individual with the agent.

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of an OAT protein (e.g., the modulation of gene expression, andor cell growth and differentiation mechanisms) can be applied not onlyin basic drug screening, but also in clinical trials. For example, theeffectiveness of an agent determined by a screening assay as describedherein to increase OAT gene expression, protein levels, or upregulateOAT activity, can be monitored in clinical trials of subjects exhibitingdecreased OAT gene expression, protein levels, or downregulated OATactivity. Alternatively, the effectiveness of an agent determined by ascreening assay to decrease OAT gene expression, protein levels, ordownregulate OAT activity, can be monitored in clinical trials ofsubjects exhibiting increased OAT gene expression, protein levels, orupregulated OAT activity. In such clinical trials, the expression oractivity of an OAT gene, and preferably, other genes that have beenimplicated in, for example, an OAT-associated disorder can be used as a“read out” or markers of the phenotype of a particular cell.

For example, and not by way of limitation, genes, including OAT, thatare modulated in cells by treatment with an agent (e.g., compound, drugor small molecule) which modulates OAT activity (e.g., identified in ascreening assay as described herein) can be identified. Thus, to studythe effect of agents on OAT-associated disorders (e.g., disorderscharacterized by deregulated organic anion transport, gene expression,and/or cell growth and differentiation mechanisms), for example, in aclinical trial, cells can be isolated and RNA prepared and analyzed forthe levels of expression of OAT and other genes implicated in theOAT-associated disorder, respectively. The levels of gene expression(e.g., a gene expression pattern) can be quantified by northern blotanalysis or RT-PCR, as described herein, or alternatively by measuringthe amount of protein produced, by one of the methods as describedherein, or by measuring the levels of activity of OAT or other genes. Inthis way, the gene expression pattern can serve as a marker, indicativeof the physiological response of the cells to the agent. Accordingly,this response state may be determined before, and at various pointsduring treatment of the individual with the agent.

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of an HST-1 polypeptide (e.g., the modulation of sugartransport) can be applied not only in basic drug screening, but also inclinical trials. For example, the effectiveness of an agent determinedby a screening assay as described herein to increase HST-1 geneexpression, polypeptide levels, or upregulate HST-l activity, can bemonitored in clinical trials of subjects exhibiting decreased HST-1 geneexpression, polypeptide levels, or downregulated HST-1 activity.Alternatively, the effectiveness of an agent determined by a screeningassay to decrease HST-1 gene expression, polypeptide levels, ordownregulate HST-1 activity, can be monitored in clinical trials ofsubjects exhibiting increased HST-1 gene expression, polypeptide levels,or upregulated HST-1 activity. In such clinical trials, the expressionor activity of an HST-1 gene, and preferably, other genes that have beenimplicated in, for example, an HST-1-associated disorder can be used asa “read out” or markers of the phenotype of a particular cell.

For example, and not by way of limitation, genes, including HST-1, thatare modulated in cells by treatment with an agent (e.g., compound, drugor small molecule) which modulates HST-1 activity (e.g., identified in ascreening assay as described herein) can be identified. Thus, to studythe effect of agents on HST-1-associated disorders (e.g., disorderscharacterized by deregulated signaling or sugar transport), for example,in a clinical trial, cells can be isolated and RNA prepared and analyzedfor the levels of expression of HST-1 and other genes implicated in theHST-1-associated disorder, respectively. The levels of gene expression(e.g., a gene expression pattern) can be quantified by northern blotanalysis or RT-PCR, as described herein, or alternatively by measuringthe amount of polypeptide produced, by one of the methods as describedherein, or by measuring the levels of activity of HST-1 or other genes.In this way, the gene expression pattern can serve as a marker,indicative of the physiological response of the cells to the agent.Accordingly, this response state may be determined before, and atvarious points during treatment of the individual with the agent.

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of a TP-2 polypeptide (e.g., the modulation of transport ofbiological molecules across membranes) can be applied not only in basicdrug screening, but also in clinical trials. For example, theeffectiveness of an agent determined by a screening assay as describedherein to increase TP-2 gene expression, polypeptide levels, orupregulate TP-2 activity, can be monitored in clinical trials ofsubjects exhibiting decreased TP-2 gene expression, polypeptide levels,or downregulated TP-2 activity. Alternatively, the effectiveness of anagent determined by a screening assay to decrease TP-2 gene expression,polypeptide levels, or downregulate TP-2 activity, can be monitored inclinical trials of subjects exhibiting increased TP-2 gene expression,polypeptide levels, or upregulated TP-2 activity. In such clinicaltrials, the expression or activity of a TP-2 gene, and preferably, othergenes that have been implicated in, for example, a TP-2-associateddisorder can be used as a “read out” or markers of the phenotype of aparticular cell.

For example, and not by way of limitation, genes, including TP-2, thatare modulated in cells by treatment with an agent (e.g., compound, drugor small molecule) which modulates TP-2 activity (e.g., identified in ascreening assay as described herein) can be identified. Thus, to studythe effect of agents on transporter-associated disorders, for example,in a clinical trial, cells can be isolated and RNA prepared and analyzedfor the levels of expression of TP-2 and other genes implicated in thetransporter-associated disorder, respectively. The levels of geneexpression (e.g., a gene expression pattern) can be quantified bynorthern blot analysis or RT-PCR, as described herein, or alternativelyby measuring the amount of polypeptide produced, by one of the methodsas described herein, or by measuring the levels of activity of TP-2 orother genes. In this way, the gene expression pattern can serve as amarker, indicative of the physiological response of the cells to theagent. Accordingly, this response state may be determined before, and atvarious points during treatment of the individual with the agent.

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of a PLTR-1 protein (e.g., the modulation of gene expression,cellular signaling, PLTR-1 activity, phospholipid transporter activity,and/or cell growth, proliferation, differentiation, absorption, and/orsecretion mechanisms) can be applied not only in basic drug screening,but also in clinical trials. For example, the effectiveness of an agentdetermined by a screening assay as described herein to increase PLTR-1gene expression, protein levels, or upregulate PLTR-1 activity, can bemonitored in clinical trials of subjects exhibiting decreased PLTR-1gene expression, protein levels, or downregulated PLTR-1 activity.Alternatively, the effectiveness of an agent determined by a screeningassay to decrease PLTR-1 gene expression, protein levels, ordownregulate PLTR-1 activity, can be monitored in clinical trials ofsubjects exhibiting increased PLTR-1 gene expression, protein levels, orupregulated PLTR-1 activity. In such clinical trials, the expression oractivity of a PLTR-1 gene, and preferably, other genes that have beenimplicated in, for example, a PLTR-1-associated disorder can be used asa “read out” or markers of the phenotype of a particular cell.

For example, and not by way of limitation, genes, including PLTR-1, thatare modulated in cells by treatment with an agent (e.g., compound, drugor small molecule) which modulates PLTR-1 activity (e.g., identified ina screening assay as described herein) can be identified. Thus, to studythe effect of agents on PLTR-1-associated disorders (e.g., disorderscharacterized by deregulated gene expression, cellular signaling, PLTR-1activity, phospholipid transporter activity, and/or cell growth,proliferation, differentiation, absorption, and/or secretionmechanisms), for example, in a clinical trial, cells can be isolated andRNA prepared and analyzed for the levels of expression of PLTR-1 andother genes implicated in the PLTR-1-associated disorder, respectively.The levels of gene expression (e.g., a gene expression pattern) can bequantified by northern blot analysis or RT-PCR, as described herein, oralternatively by measuring the amount of protein produced, by one of themethods as described herein, or by measuring the levels of activity ofPLTR-1 or other genes. In this way, the gene expression pattern canserve as a marker, indicative of the physiological response of the cellsto the agent. Accordingly, this response state may be determined before,and at various points during treatment of the individual with the agent.

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of a TFM-2 and/or TFM-3 polypeptide (e.g., the modulation oftransport of biological molecules across membranes) can be applied notonly in basic drug screening, but also in clinical trials. For example,the effectiveness of an agent determined by a screening assay asdescribed herein to increase TFM-2 and/or TFM-3 gene expression,polypeptide levels, or upregulate TFM-2 and/or TFM-3 activity, can bemonitored in clinical trials of subjects exhibiting decreased TFM-2and/or TFM-3 gene expression, polypeptide levels, or downregulated TFM-2and/or TFM-3 activity. Alternatively, the effectiveness of an agentdetermined by a screening assay to decrease TFM-2 and/or TFM-3 geneexpression, polypeptide levels, or downregulate TFM-2 and/or TFM-3activity, can be monitored in clinical trials of subjects exhibitingincreased TFM-2 and/or TFM-3 gene expression, polypeptide levels, orupregulated TFM-2 and/or TFM-3 activity. In such clinical trials, theexpression or activity of a TFM-2 and/or TFM-3 gene, and preferably,other genes that have been implicated in, for example, a TFM-2 and/orTFM-3-associated disorder can be used as a “read out” or markers of thephenotype of a particular cell.

For example, and not by way of limitation, genes, including TFM-2 and/orTFM-3, that are modulated in cells by treatment with an agent (e.g.,compound, drug or small molecule) which modulates TFM-2 and/or TFM-3activity (e.g., identified in a screening assay as described herein) canbe identified. Thus, to study the effect of agents ontransporter-associated disorders, for example, in a clinical trial,cells can be isolated and RNA prepared and analyzed for the levels ofexpression of TFM-2 and/or TFM-3 and other genes implicated in thetransporter-associated disorder, respectively. The levels of geneexpression (e.g., a gene expression pattern) can be quantified bynorthern blot analysis or RT-PCR, as described herein, or alternativelyby measuring the amount of polypeptide produced, by one of the methodsas described herein, or by measuring the levels of activity of TFM-2and/or TFM-3 or other genes. In this way, the gene expression patterncan serve as a marker, indicative of the physiological response of thecells to the agent. Accordingly, this response state may be determinedbefore, and at various points during treatment of the individual withthe agent.

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of a 67118, 67067, and/or 62092 polypeptide (e.g., themodulation of gene expression, cellular signaling, 67118, 67067, and/or62092 activity, phospholipid transporter activity, and/or cell growth,proliferation, differentiation, absorption, and/or secretion mechanisms)can be applied not only in basic drug screening, but also in clinicaltrials. For example, the effectiveness of an agent determined by ascreening assay as described herein to increase 67118, 67067, and/or62092 gene expression, polypeptide levels, or upregulate 67118, 67067,and/or 62092 activity, can be monitored in clinical trials of subjectsexhibiting decreased 67118, 67067, and/or 62092 gene expression,polypeptide levels, or downregulated 67118, 67067, and/or 62092activity. Alternatively, the effectiveness of an agent determined by ascreening assay to decrease 67118, 67067, and/or 62092 gene expression,polypeptide levels, or downregulate 67118, 67067, and/or 62092 activity,can be monitored in clinical trials of subjects exhibiting increased67118, 67067, and/or 62092 gene expression, polypeptide levels, orupregulated 67118, 67067, and/or 62092 activity. In such clinicaltrials, the expression or activity of a 67118, 67067, and/or 62092 gene,and preferably, other genes that have been implicated in, for example, a67118, 67067, and/or 62092-associated disorder can be used as a “readout” or markers of the phenotype of a particular cell.

For example, and not by way of limitation, genes, including 67118,67067, and/or 62092, that are modulated in cells by treatment with anagent (e.g., compound, drug or small molecule) which modulates 67118,67067, and/or 62092 activity (e.g., identified in a screening assay asdescribed herein) can be identified. Thus, to study the effect of agentson 67118, 67067, or 62092-associated disorders (e.g., disorderscharacterized by deregulated gene expression, cellular signaling, 67118or 67067 activity, phospholipid transporter activity, and/or cellgrowth, proliferation, differentiation, absorption, and/or secretionmechanisms or disorders characterized by 62092 activity, nucleotidebinding activity, and/or apoptosis mechanisms), for example, in aclinical trial, cells can be isolated and RNA prepared and analyzed forthe levels of expression of 67118, 67067, and/or 62092 and other genesimplicated in the 67118, 67067, or 62092-associated disorder,respectively. The levels of gene expression (e.g., a gene expressionpattern) can be quantified by northern blot analysis or RT-PCR, asdescribed herein, or alternatively by measuring the amount ofpolypeptide produced, by one of the methods as described herein, or bymeasuring the levels of activity of 67118, 67067, and/or 62092 or othergenes. In this way, the gene expression pattern can serve as a marker,indicative of the physiological response of the cells to the agent.Accordingly, this response state may be determined before, and atvarious points during treatment of the individual with the agent.

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of a HAAT protein (e.g., the modulation of protein synthesis,hormone metabolism, nerve transmission, cellular activation, regulationof cell growth, production of metabolic energy, synthesis of purines andpyrimidines, nitrogen metabolism, and/or biosynthesis of urea) can beapplied not only in basic drug screening, but also in clinical trials.For example, the effectiveness of an agent determined by a screeningassay as described herein to increase HAAT gene expression, proteinlevels, or upregulate HAAT activity, can be monitored in clinical trialsof subjects exhibiting decreased HAAT gene expression, protein levels,or downregulated HAAT activity. Alternatively, the effectiveness of anagent determined by a screening assay to decrease HAAT gene expression,protein levels, or downregulate HAAT activity, can be monitored inclinical trials of subjects exhibiting increased HAAT gene expression,protein levels, or upregulated HAAT activity. In such clinical trials,the expression or activity of a HAAT gene, and preferably, other genesthat have been implicated in, for example, a HAAT-associated disordercan be used as a “read out” or markers of the phenotype of a particularcell.

For example, and not by way of limitation, genes, including HAAT, thatare modulated in cells by treatment with an agent (e.g., compound, drugor small molecule) which modulates HAAT activity (e.g., identified in ascreening assay as described herein) can be identified. Thus, to studythe effect of agents on HAAT-associated disorders (e.g., disorderscharacterized by deregulated protein synthesis, hormone metabolism,nerve transmission, cellular activation, regulation of cell growth,production of metabolic energy, synthesis of purines and pyrimidines,nitrogen metabolism, and/or biosynthesis of urea), for example, in aclinical trial, cells can be isolated and RNA prepared and analyzed forthe levels of expression of HAAT and other genes implicated in theHAAT-associated disorder, respectively. The levels of gene expression(e.g., a gene expression pattern) can be quantified by northern blotanalysis or RT-PCR, as described herein, or alternatively by measuringthe amount of protein produced, by one of the methods as describedherein, or by measuring the levels of activity of HAAT or other genes.In this way, the gene expression pattern can serve as a marker,indicative of the physiological response of the cells to the agent.Accordingly, this response state may be determined before, and atvarious points during treatment of the individual with the agent.

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of an HST-4 and/or an HST-5 polypeptide (e.g., the modulationof sugar transport) can be applied not only in basic drug screening, butalso in clinical trials. For example, the effectiveness of an agentdetermined by a screening assay as described herein to increase HST-4and/or HST-5 gene expression, polypeptide levels, or upregulate HST-4and/or HST-5 activity, can be monitored in clinical trials of subjectsexhibiting decreased HST-4 and/or HST-5 gene expression, polypeptidelevels, or downregulated HST-4 and/or HST-5 activity. Alternatively, theeffectiveness of an agent determined by a screening assay to decreaseHST-4 and/or HST-5 gene expression, polypeptide levels, or downregulateHST-4 and/or HST-5 activity, can be monitored in clinical trials ofsubjects exhibiting increased HST-4 and/or HST-5 gene expression,polypeptide levels, or upregulated HST-4 and/or HST-5 activity. In suchclinical trials, the expression or activity of an HST-4 and/or HST-5gene, and preferably, other genes that have been implicated in, forexample, an HST-4- and/or an HST-5-associated disorder can be used as a“read out” or markers of the phenotype of a particular cell.

For example, and not by way of limitation, genes, including HST-4 and/orHST-5, that are modulated in cells by treatment with an agent (e.g.,compound, drug or small molecule) which modulates HST-4 and/or HST-5activity (e.g., identified in a screening assay as described herein) canbe identified. Thus, to study the effect of agents on HST-4- and/orHST-5-associated disorders (e.g., disorders characterized by deregulatedsignaling or sugar transport), for example, in a clinical trial, cellscan be isolated and RNA prepared and analyzed for the levels ofexpression of HST-4 and/or HST-5 and other genes implicated in theHST-4- and/or the HST-5-associated disorder, respectively. The levels ofgene expression (e.g., a gene expression pattern) can be quantified bynorthern blot analysis or RT-PCR, as described herein, or alternativelyby measuring the amount of polypeptide produced, by one of the methodsas described herein, or by measuring the levels of activity of HST-4and/or HST-5 or other genes. In this way, the gene expression patterncan serve as a marker, indicative of the physiological response of thecells to the agent. Accordingly, this response state may be determinedbefore, and at various points during treatment of the individual withthe agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) including the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of an MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 polypeptide, mRNA, or genomic DNA in the preadministrationsample; (iii) obtaining one or more post-administration samples from thesubject; (iv) detecting the level of expression or activity of the HST-4and/or the HST-5 polypeptide, mRNA, or genomic DNA in thepost-administration samples; (v) comparing the level of expression oractivity of the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or the HST-5 polypeptide, mRNA, or genomicDNA in the pre-administration sample with the MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or the HST-5polypeptide, mRNA, or genomic DNA in the post administration sample orsamples; and (vi) altering the administration of the agent to thesubject accordingly. For example, increased administration of the agentmay be desirable to increase the expression or activity of MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 to higher levels than detected, i.e., to increase theeffectiveness of the agent. Alternatively, decreased administration ofthe agent may be desirable to decrease expression or activity of MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 to lower levels than detected, i.e. to decrease theeffectiveness of the agent. According to such an embodiment, MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 expression or activity may be used as an indicator of theeffectiveness of an agent, even in the absence of an observablephenotypic response.

E. Methods of Treatment:

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant or unwanted MTP-1expression or activity, e.g., a transporter-associated disorder such asa hematopoietic disorder, an immunological disorder, a lipidmetabolism-related disorder, a CNS disorder; a cellular proliferation,growth, differentiation, or migration disorder; a, musculoskeletaldisorder; a cardiovascular disorder; an immune disorder; or a hormonaldisorder. The present invention provides for both prophylactic andtherapeutic methods of treating a subject at risk of (or susceptible to)a disorder or having an OAT-associated disorder, e.g., a disorderassociated with aberrant or unwanted OAT expression or activity. Thepresent invention provides for both prophylactic and therapeutic methodsof treating a subject at risk of (or susceptible to) a disorder orhaving a disorder associated with aberrant or unwanted HST-1 expressionor activity, e.g. a sugar transporter disorder. The present inventionprovides for both prophylactic and therapeutic methods of treating asubject at risk of (or susceptible to) a disorder or having a disorderassociated with aberrant or unwanted TP-2 expression or activity, e.g. atransporter-associated disorder. The present invention provides for bothprophylactic and therapeutic methods of treating a subject at risk of(or susceptible to) a disorder or having a PLTR-1-associated disorder,e.g., a disorder associated with aberrant or unwanted PLTR-1 expressionor activity (e.g., a cardiovascular disorder). The present inventionprovides for both prophylactic and therapeutic methods of treating asubject at risk of (or susceptible to) a disorder or having a disorderassociated with aberrant or unwanted TFM-2 and/or TFM-3 expression oractivity, e.g. a transporter-associated disorder. The present inventionprovides for both prophylactic and therapeutic methods of treating asubject at risk of (or susceptible to) a disorder or having a disorderassociated with aberrant or unwanted 67118, 67067, and/or 62092expression or activity, e.g. a phospholipid transporter-associateddisorder. The present invention provides for both prophylactic andtherapeutic methods of treating a subject at risk of (or susceptible to)a disorder or having a HAAT-associated disorder, e.g., a disorderassociated with aberrant or unwanted HAAT expression or activity. Thepresent invention provides for both prophylactic and therapeutic methodsof treating a subject at risk of (or susceptible to) a disorder orhaving a disorder associated with aberrant or unwanted HST-4 and/orHST-5 expression or activity, e.g. a sugar transporter disorder.“Treatment”, as used herein, is defined as the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell froma patient, who has a disease or disorder, a symptom of disease ordisorder or a predisposition toward a disease or disorder, or is at riskof (or susceptible to) a disease or disorder, with the purpose to cure,heal, alleviate, relieve, alter, remedy, ameliorate, improve or affectthe disease or disorder, the symptoms of disease or disorder, the riskof (or susceptibility to) the disease or disorder or the predispositiontoward a disease or disorder. A therapeutic agent includes, but is notlimited to, small molecules, peptides, polypeptides, antibodies,ribozymes and antisense oligonucleotides.

With regards to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or theHST-5 molecules of the present invention or MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5modulators according to that individual's drug response genotype.Pharmacogenomics allows a clinician or physician to target prophylacticor therapeutic treatments to patients who will most benefit from thetreatment and to avoid treatment of patients who will experience toxicdrug-related side effects.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant or unwantedMTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5 expression or activity, by administering to thesubject an MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 or an agent which modulates MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 expression or at least one MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 activity.Subjects at risk for a disease which is caused or contributed to byaberrant or unwanted MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5 expression or activity canbe identified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5 aberrancy, such that a disease or disorder isprevented or, alternatively, delayed in its progression. Depending onthe type of MTP-1, OAT, HST-1, TP-2, PLTR-1,-TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 aberrancy, for example, MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 agonist or MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5 antagonist agent can beused for treating the subject. The appropriate agent can be determinedbased on screening assays described herein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 expression or activity for therapeutic purposes.Accordingly, in an exemplary embodiment, the modulatory method of theinvention involves contacting a cell capable of expressing MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 with an agent that modulates one or more of the activitiesof MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5 polypeptide activity associated with the cell,such that MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 activity in the cell is modulated. Anagent that modulates MTP-1 protein activity can be an agent as describedherein, such as a nucleic acid or a protein, a naturally-occurringsubstrate molecule of an MTP-1 protein (e.g., cytotoxic substances,ions, peptides, metabolites), an MTP-1 antibody, an MTP-1 agonist orantagonist, a peptidomimetic of an MTP-1 agonist or antagonist, or othersmall molecule. An agent that modulates OAT protein activity can be anagent as described herein, such as a nucleic acid or a protein, anaturally-occurring target molecule of an OAT protein (e.g., an OATsubstrate), an OAT antibody, an OAT agonist or antagonist, apeptidomimetic of an OAT agonist or antagonist, or other small molecule.An agent that modulates HST-1 polypeptide activity can be an agent asdescribed herein, such as a nucleic acid or a polypeptide, anaturally-occurring target molecule of an HST-1 polypeptide (e.g., anHST-1 substrate), an HST-1 antibody, an HST-1 agonist or antagonist, apeptidomimetic of an HST-1 agonist or antagonist, or other smallmolecule. An agent that modulates TP-2 polypeptide activity can be anagent as described herein, such as a nucleic acid or a polypeptide, anaturally-occurring target molecule of a TP-2 polypeptide (e.g., a TP-2substrate), a TP-2 antibody, a TP-2 agonist or antagonist, apeptidomimetic of a TP-2 agonist or antagonist, or other small molecule.An agent that modulates PLTR-1 protein activity can be an agent asdescribed herein, such as a nucleic acid or a protein, anaturally-occurring target molecule of a PLTR-1 protein (e.g., a PLTR-1substrate), a PLTR-1 antibody, a PLTR-1 agonist or antagonist, apeptidomimetic of a PLTR-1 agonist or antagonist, or other smallmolecule. An agent that modulates TFM-2 and/or TFM-3 polypeptideactivity can be an agent as described herein, such as a nucleic acid ora polypeptide, a naturally-occurring target molecule of a TFM-2 and/orTFM-3 polypeptide (e.g., a TFM-2 and/or TFM-3 substrate), a TFM-2 and/orTFM-3 antibody, a TFM-2 and/or TFM-3 agonist or antagonist, apeptidomimetic of a TFM-2 and/or TFM-3 agonist or antagonist, or othersmall molecule. An agent that modulates 67118, 67067, and/or 62092polypeptide activity can be an agent as described herein, such as anucleic acid or a polypeptide, a naturally-occurring target molecule ofa 67118, 67067, and/or 62092 polypeptide (e.g., a 67118, 67067, and/or62092 substrate), a 67118, 67067, and/or 62092 antibody, a 67118, 67067,and/or 62092 agonist or antagonist, a peptidomimetic of a 67118, 67067,and/or 62092 agonist or antagonist, or other small molecule. An agentthat modulates HAAT protein activity can be an agent as describedherein, such as a nucleic acid or a protein, a naturally-occurringtarget molecule of a HAAT protein (e.g., a HAAT substrate), a HAATantibody, a HAAT agonist or antagonist, a peptidomimetic of a HAATagonist or antagonist, or other small molecule. An agent that modulatesHST-4 and/or HST-5 polypeptide activity can be an agent as describedherein, such as a nucleic acid or a polypeptide, a naturally-occurringtarget molecule of an HST-4 and/or an HST-5 polypeptide (e.g., an HST-4and/or an HST-5 substrate), an HST-4 and/or an HST-5 antibody, an HST-4and/or an HST-5 agonist or antagonist, a peptidomimetic of an HST-4and/or an HST-5 agonist or antagonist, or other small molecule. In oneembodiment, the agent stimulates one or more MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5activities. Examples of such stimulatory agents include active MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 polypeptides and nucleic acid molecules encoding MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 that have been introduced into the cell. In anotherembodiment, the agent inhibits one or more MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5activities. Examples of such inhibitory agents include antisense MTP-1,OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 nucleic acid molecules, anti-HST-4 and/or -HST-5antibodies, and MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 inhibitors. These modulatorymethods can be performed in vitro (e.g., by culturing the cell with theagent) or, alternatively, in vivo (e.g., by administering the agent to asubject). As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant or unwanted expression or activity of an MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or anHST-5 polypeptide or nucleic acid molecule. In one embodiment, themethod involves administering an agent (e.g., an agent identified by ascreening assay described herein), or combination of agents thatmodulates (e.g., upregulates or downregulates) MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5expression or activity. In another embodiment, the method involvesadministering an MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or an HST-5 polypeptide or nucleic acidmolecule as therapy to compensate for reduced, aberrant, or unwantedMTP-1, OAT, HST-1, TP-2, PLTR-1,-TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5 expression or activity.

Stimulation of MTP-1 activity is desirable in situations in which MTP-1is abnormally downregulated and/or in which increased MTP-1 activity islikely to have a beneficial effect. Likewise, inhibition of MTP-1activity is desirable in situations in which MTP-1 is abnormallyupregulated and/or in which decreased MTP-1 activity is likely to have abeneficial effect.

(i) Methods for Inhibiting Target Gene MTP-1 Expression, Synthesis, orActivity

As discussed above, genes involved in hematopoietic and/or immunologicaland/or lipid metabolism-related diseases or disorders may cause suchdisorders via an increased level of gene activity. In some cases, suchup-regulation may have a causative or exacerbating effect on the diseasestate. A variety of techniques may be used to inhibit the expression,synthesis, or activity of such genes and/or proteins.

For example, compounds such as those identified through assays describedabove, which exhibit inhibitory activity, may be used in accordance withthe invention to ameliorate hematopoietic and/or immunological and/orlipid metabolism-related disease or disorder symptoms. Such moleculesmay include, but are not limited to, small organic molecules, peptides,antibodies, and the like.

For example, compounds can be administered that compete with endogenousligand for the MTP-1 protein. The resulting reduction in the amount ofligand-bound MTP-1 protein will modulate endothelial cell physiology.Compounds that can be particularly useful for this purpose include, forexample, soluble proteins or peptides, such as peptides comprising oneor more of the extra-membrane domains, or portions and/or analogsthereof, of the MTP-1 protein, including, for example, soluble fusionproteins such as Ig-tailed fusion proteins. (For a discussion of theproduction of Ig-tailed fusion proteins, see, for example, U.S. Pat. No.5,116,964). Alternatively, compounds, such as ligand analogs orantibodies, that bind to the MTP-1 active site, but do not activate theprotein, can be effective in inhibiting MTP-1 protein activity.

Further, antisense and ribozyme molecules, as described herein, whichinhibit expression of the MTP-1 gene may also be used in accordance withthe invention to inhibit aberrant MTP-1 gene activity. Still further,triple helix molecules may be utilized in inhibiting aberrant MTP-1 geneactivity.

Antibodies that are both specific for the MTP-1 protein and interferewith its activity may also be used to modulate or inhibit MTP-1 proteinfunction. Such antibodies may be generated using standard techniquesdescribed herein, against the MTP-1 protein itself or against peptidescorresponding to portions of the protein. Such antibodies include butare not limited to polyclonal, monoclonal, Fab fragments, single chainantibodies, or chimeric antibodies.

In instances where the target gene protein is intracellular and wholeantibodies are used, internalizing antibodies may be preferred.Lipofectin liposomes may be used to deliver the antibody or a fragmentof the Fab region which binds to the target epitope into cells. Wherefragments of the antibody are used, the smallest inhibitory

fragment which binds to the target protein's binding domain ispreferred. For example, peptides having an amino acid sequencecorresponding to the domain of the variable region of the antibody thatbinds to the target gene protein may be used. Such peptides may besynthesized chemically or produced via recombinant DNA technology using

methods well known in the art (described in, for example, Creighton(1983), supra; and Sambrook et al. (1989) supra). Single chainneutralizing antibodies which bind to intracellular target gene epitopesmay also be administered. Such single chain antibodies may beadministered, for example, by expressing nucleotide sequences encodingsingle-chain antibodies within the target cell population by utilizing,for example, techniques such as those described in Marasco et al. (1993)Proc. Natl. Acad. Sci. USA 90:7889-7893).

Any of the administration techniques described below which areappropriate for peptide administration may be utilized to effectivelyadminister inhibitory target gene antibodies to their site of action.

(ii) Methods for Restoring or Enhancing Target Gene MTP-1 Activity

Genes that cause hematopoietic and/or immunological and/or lipidmetabolism-related diseases or disorders may be underexpressed withincellular growth or proliferative situations. Alternatively, the activityof the protein products of such genes may be decreased, leading to thedevelopment of hematopoietic and/or immunological and/or lipidmetabolism-related disease or disorder symptoms. Such down-regulation ofgene expression or decrease of protein activity might have a causativeor exacerbating effect on the disease state.

In some cases, genes that are up-regulated in the disease state might beexerting a protective effect. A variety of techniques may be used toincrease the expression, synthesis, or activity of genes and/or proteinsthat exert a protective effect in response to hematopoietic and/orimmunological and/or lipid metabolism-related disease or disorderconditions.

Described in this section are methods whereby the level MTP-1 activitymay be, increased to levels wherein hematopoietic and/or immunologicaland/or lipid metabolism-related disease or disorder symptoms areameliorated. The level of MTP-1 activity may be increased, for example,by either increasing the level of MTP-1 gene expression or by increasingthe level of active MTP-1 protein which is present.

For example, a MTP-1 protein, at a level sufficient to amelioratehematopoietic and/or immunological and/or lipid metabolism-relateddisease or disorder symptoms may be administered to a patient exhibitingsuch symptoms. Any of the techniques discussed below may be used forsuch administration. One of skill in the art will readily be able toascertain the concentration of effective, non-toxic doses of the MTP-1protein, utilizing techniques such as those described above.

Additionally, RNA sequences encoding a MTP-1 protein may be directlyadministered to a patient exhibiting hematopoietic and/or immunologicaland/or lipid metabolism-related disease or disorder symptoms, at aconcentration sufficient to produce a level of MTP-1 protein such thathematopoietic and/or immunological and/or lipid metabolism-relateddisease or disorder symptoms are ameliorated. Any of the techniquesdiscussed below, which achieve intracellular administration ofcompounds, such as, for example, liposome administration, may be usedfor the administration of such RNA molecules. The RNA molecules may beproduced, for example, by recombinant techniques such as those describedherein.

Further, subjects may be treated by gene replacement therapy. One ormore copies of a MTP-1 gene, or a portion thereof, that directs theproduction of a normal MTP-1 protein with MTP-1 function, may beinserted into cells using vectors which include, but are not limited toadenovirus, adeno-associated virus, and retrovirus vectors, in additionto other particles that introduce DNA into cells, such as liposomes.Additionally, techniques such as those described above may be used forthe introduction of MTP-1 gene sequences into human cells.

Cells, preferably, autologous cells, containing MTP-1 expressing genesequences may then be introduced or reintroduced into the subject atpositions which allow for the amelioration of hematopoietic and/orimmunological and/or lipid metabolism-related disease or disordersymptoms. Such cell replacement techniques may be preferred, forexample, when the gene product is a secreted, extracellular geneproduct.

Stimulation of OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 activity is desirable in situations inwhich OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT,HST-4 and/or HST-5 is abnormally downregulated and/or in which increasedOAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 activity is likely to have a beneficial effect. Likewise,inhibition of OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 activity is desirable in situations inwhich OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT,HST-4 and/or HST-5 is abnormally upregulated and/or in which decreasedOAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 activity is likely to have a beneficial effect.

3. Pharmacogenomics

The MTP-1 molecules of the present invention, as well as agents, ormodulators which have a stimulatory or inhibitory effect on MTP-1activity (e.g., MTP-1 gene expression) as identified by a screeningassay described herein can be administered to individuals to treat(prophylactically or therapeutically) MTP-1-associated disorders (e.g.,proliferative disorders, CNS disorders, cardiac disorders, metabolicdisorders, or muscular disorders) associated with aberrant or unwantedMTP-1 activity. In conjunction with such treatment, pharmacogenomics(i.e., the study of the relationship between an individual's genotypeand that individual's response to a foreign compound or drug) may beconsidered. Differences in metabolism of therapeutics can lead to severetoxicity or therapeutic failure by altering the relation between doseand blood concentration of the pharmacologically active drug. Thus, aphysician or clinician may consider applying knowledge obtained inrelevant pharmacogenomics studies in determining whether to administeran MTP-1 molecule or MTP-1 modulator as well as tailoring the dosageand/or therapeutic regimen of treatment with an MTP-1 molecule or MTP-1modulator.

The OAT molecules of the present invention, as well as agents, ormodulators which have a stimulatory or inhibitory effect on OAT activity(e.g., OAT gene expression) as identified by a screening assay describedherein can be administered to individuals to treat (prophylactically ortherapeutically) OAT-associated disorders (e.g., disorders characterizedby aberrant organic anion transport, and/or gene expression, CNS,cardiac, musculoskeletal, metabolic, cell proliferation and/ordifferentiation disorders) associated with aberrant or unwanted OATactivity. In conjunction with such treatment, pharmacogenomics (i.e.,the study of the relationship between an individual's genotype and thatindividual's response to a foreign compound or drug) may be considered.Differences in metabolism of therapeutics can lead to severe toxicity ortherapeutic failure by altering the relation between dose and bloodconcentration of the pharmacologically active drug. Thus, a physician orclinician may consider applying knowledge obtained in relevantpharmacogenomics studies in determining whether to administer an OATmolecule or OAT modulator as well as tailoring the dosage and/ortherapeutic regimen of treatment with an OAT molecule or OAT modulator.

The HST-1 molecules of the present invention, as well as agents, ormodulators which have a stimulatory or inhibitory effect on HST-1activity (e.g., HST-1 gene expression) as identified by a screeningassay described herein can be administered to individuals to treat(prophylactically or therapeutically) HST-1-associated disorders (e.g.,proliferative disorders) associated with aberrant or unwanted HST-1activity. In conjunction with such treatment, pharmacogenomics (i.e.,the study of the relationship between an individual's genotype and thatindividual's response to a foreign compound or drug) may be considered.Differences in metabolism of therapeutics can lead to severe toxicity ortherapeutic failure by altering the relation between dose and bloodconcentration of the pharmacologically active drug. Thus, a physician orclinician may consider applying knowledge obtained in relevantpharmacogenomics studies in determining whether to administer an HST-1molecule or HST-1 modulator as well as tailoring the dosage and/ortherapeutic regimen of treatment with an HST-1 molecule or HST-1modulator.

The TP-2 molecules of the present invention, as well as agents, ormodulators which have a stimulatory or inhibitory effect on TP-2activity (e.g., TP-2 gene expression) as identified by a screening assaydescribed herein can be administered to individuals to treat(prophylactically or therapeutically) transporter-associated disorders(e.g., proliferative disorders) associated with aberrant or unwantedTP-2 activity. In conjunction with such treatment, pharmacogenomics(i.e., the study of the relationship between an individual's genotypeand that individual's response to a foreign compound or drug) may beconsidered. Differences in metabolism of therapeutics can lead to severetoxicity or therapeutic failure by altering the relation between doseand blood concentration of the pharmacologically active drug. Thus, aphysician or clinician may consider applying knowledge obtained inrelevant pharmacogenomics studies in determining whether to administer aTP-2 molecule or TP-2 modulator as well as tailoring the dosage and/ortherapeutic regimen of treatment with a TP-2 molecule or TP-2 modulator.

The PLTR-1 molecules of the present invention, as well as agents, ormodulators which have a stimulatory or inhibitory effect on PLTR-1activity (e.g., PLTR-1 gene expression) as identified by a screeningassay described herein can be administered to individuals to treat(prophylactically or therapeutically) PLTR-1-associated disorders (e.g.,disorders characterized by aberrant gene expression, PLTR-1 activity,phospholipid transporter activity, cellular signaling, and/or cellgrowth, proliferation, differentiation, absorption, and/or secretion)associated with aberrant or unwanted PLTR-1 activity. In conjunctionwith such treatment, pharmacogenomics (i.e., the study of therelationship between an individual's genotype and that individual'sresponse to a foreign compound or drug) may be considered. Differencesin metabolism of therapeutics can lead to severe toxicity or therapeuticfailure by altering the relation between dose and blood concentration ofthe pharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a PLTR-1 molecule or PLTR-1modulator as well as tailoring the dosage and/or therapeutic regimen oftreatment with a PLTR-1 molecule or PLTR-1 modulator.

The TFM-2 and/or TFM-3 molecules of the present invention, as well asagents, or modulators which have a stimulatory or inhibitory effect onTFM-2 and/or TFM-3 activity (e.g., TFM-2 and/or TFM-3 gene expression)as identified by a screening assay described herein can be administeredto individuals to treat (prophylactically or therapeutically)transporter-associated disorders (e.g., proliferative disorders)associated with aberrant or unwanted TFM-2 and/or TFM-3 activity. Inconjunction with such treatment, pharmacogenomics (i.e., the study ofthe relationship between an individual's genotype and that individual'sresponse to a foreign compound or drug) may be considered. Differencesin metabolism of therapeutics can lead to severe toxicity or therapeuticfailure by altering the relation between dose and blood concentration ofthe pharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a TFM-2 and/or TFM-3molecule or TFM-2 and/or TFM-3 modulator as well as tailoring the dosageand/or therapeutic regimen of treatment with a TFM-2 and/or TFM-3molecule or TFM-2 and/or TFM-3 modulator.

The 67118, 67067, and/or 62092 molecules of the present invention, aswell as agents, or modulators which have a stimulatory or inhibitoryeffect on 67118, 67067, and/or 62092 activity (e.g., 67118, 67067,and/or 62092 gene expression) as identified by a screening assaydescribed herein can be administered to individuals to treat(prophylactically or therapeutically) 67118, 67067, or 62092-associateddisorders (e.g., disorders characterized by aberrant gene expression,67118, 67067, and/or 62092 activity, phospholipid transporter activity,cellular signaling, and/or cell growth, proliferation, differentiation,absorption, and/or secretion disorders or disorders characterized by62092 activity, nucleotide binding activity, and/or apoptosismechanisms) associated with aberrant or unwanted 67118, 67067, and/or62092 activity. In conjunction with such treatment, pharmacogenomics(i.e., the study of the relationship between an individual's genotypeand that individual's response to a foreign compound or drug) may beconsidered. Differences in metabolism of therapeutics can lead to severetoxicity or therapeutic failure by altering the relation between doseand blood concentration of the pharmacologically active drug. Thus, aphysician or clinician may consider applying knowledge obtained inrelevant pharmacogenomics studies in determining whether to administer a67118, 67067, and/or 62092 molecule or 67118, 67067, and/or 62092modulator as well as tailoring the dosage and/or therapeutic regimen oftreatment with a 67118, 67067, and/or 62092 molecule or 67118, 67067,and/or 62092 modulator.

The HAAT molecules of the present invention, as well as agents, ormodulators which have a stimulatory or inhibitory effect on HAATactivity (e.g., HAAT gene expression) as identified by a screening assaydescribed herein can be administered to individuals to treat(prophylactically or therapeutically) HAAT-associated disorders (e.g.,disorders characterized by aberrant protein synthesis, hormonemetabolism, nerve transmission, cellular activation, regulation of cellgrowth, production of metabolic energy, synthesis of purines andpyrimidines, nitrogen metabolism, and/or biosynthesis of urea)associated with aberrant or unwanted HAAT activity. In conjunction withsuch treatment, pharmacogenomics (i.e., the study of the relationshipbetween an individual's genotype and that individual's response to aforeign compound or drug) may be considered. Differences in metabolismof therapeutics can lead to severe toxicity or therapeutic failure byaltering the relation between dose and blood concentration of thepharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a HAAT molecule or HAATmodulator as well as tailoring the dosage and/or therapeutic regimen oftreatment with a HAAT molecule or HAAT modulator.

The HST-4 and/or HST-5 molecules of the present invention, as well asagents, or modulators which have a stimulatory or inhibitory effect onHST-4 and/or HST-5 activity (e.g., HST-4 and/or HST-5 gene expression)as identified by a screening assay described herein can be administeredto individuals to treat (prophylactically or therapeutically) HST-4-and/or HST-5-associated disorders (e.g., proliferative disorders)associated with aberrant or unwanted HST-4 and/or HST-5 activity. Inconjunction with such treatment, pharmacogenomics (i.e., the study ofthe relationship between an individual's genotype and that individual'sresponse to a foreign compound or drug) may be considered. Differencesin metabolism of therapeutics can lead to severe toxicity or therapeuticfailure by altering the relation between dose and blood concentration ofthe pharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer an HST-4 molecule and/or anHST-5 molecule or an HST-4 modulator and/or an HST-5 modulator as wellas tailoring the dosage and/or therapeutic regimen of treatment with anHST-4 molecule and/or an HST-5 molecule or an HST-4 modulator and/or anHST-5 modulator.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, for example, Eichelbaum, M. et al.(1996) Clin. Exp. Pharmacol. Physiol. 23(10-11): 983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach”, can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drugs target is known (e.g., an MTP-1,an OAT, an HST-1, a TP-2, a PLTR-1, a TFM-2, a TFM-3, a 67118, a 67067,a 62092, a HAAT, an HST-4 and/or an HST-5 polypeptide of the presentinvention), all common variants of that gene can be fairly easilyidentified in the population and it can be determined if having oneversion of the gene versus another is associated with a particular drugresponse.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Alternatively, a method termed the “gene expression profiling”, can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., an MTP-1, an OAT,an HST-1, a TP-2, a PLTR-1, a TFM-2, a TFM-3, a 67118, a 67067, a 62092,a HAAT, an HST-4 molecule and/or an HST-5 molecule or an MTP-1, an OAT,an HST-1, a TP-2, a PLTR-1, a TFM-2, a TFM-3, a 67118, a 67067, a 62092,a HAAT, an HST-4 modulator and/or an HST-5 modulator of the presentinvention) can give an indication whether gene pathways related totoxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment an individual. Thisknowledge, when applied to dosing or drug selection, can avoid adversereactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with an MTP-1, an OAT, nHST-1, a TP-2, a PLTR-1, a TFM-2, a TFM-3, a 67118, a 67067, a 62092, aHAAT, an HST-4 and/or an HST-5 molecule or an MTP-1, an OAT, an HST-1, aTP-2, a PLTR-1, a TFM-2, a TFM-3, a 67118, a 67067, a 62092, a HAAT, anHST-4 and/or an HST-5 modulator, such as a modulator identified by oneof the exemplary screening assays described herein.

4. Use of MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067,62092, HAAT, HST-4 and HST-5 Molecules as Surrogate Markers

The MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and HST-5 molecules of the invention are also useful asmarkers of disorders or disease states, as markers for precursors ofdisease states, as markers for predisposition of disease states, asmarkers of drug activity, or as markers of the pharmacogenomic profileof a subject. Using the methods described herein, the presence, absenceand/or quantity of the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or the HST-5 molecules of theinvention may be detected, and may be correlated with one or morebiological states in vivo. For example, the MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or the HST-5molecules of the invention may serve as surrogate markers for one ormore disorders or disease states or for conditions leading up to diseasestates. As used herein, a “surrogate marker” is an objective biochemicalmarker which correlates with the absence or presence of a disease ordisorder, or with the progression of a disease or disorder (e.g., withthe presence or absence of a tumor). The presence or quantity of suchmarkers is independent of the disease. Therefore, these markers mayserve to indicate whether a particular course of treatment is effectivein lessening a disease state or disorder. Surrogate markers are ofparticular use when the presence or extent of a disease state ordisorder is difficult to assess through standard methodologies (e.g.,early stage tumors), or when an assessment of disease progression isdesired before a potentially dangerous clinical endpoint is reached(e.g., an assessment of cardiovascular disease may be made usingcholesterol levels as a surrogate marker, and an analysis of HIVinfection may be made using HIV RNA levels as a surrogate marker, wellin advance of the undesirable clinical outcomes of myocardial infarctionor fully-developed AIDS). Examples of the use of surrogate markers inthe art include: Koomen et al. (2000) J. Mass. Spectrom. 35: 258-264;and James (1994) AIDS Treatment News Archive 209.

The MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or the HST-5 molecules of the invention are also usefulas pharmacodynamic markers. As used herein, a “pharmacodynamic marker”is an objective biochemical marker which correlates specifically withdrug effects. The presence or quantity of a pharmacodynamic marker isnot related to the disease state or disorder for which the drug is beingadministered; therefore, the presence or quantity of the marker isindicative of the presence or activity of the drug in a subject. Forexample, a pharmacodynamic marker may be indicative of the concentrationof the drug in a biological tissue, in that the marker is eitherexpressed or transcribed or not expressed or transcribed in that tissuein relationship to the level of the drug. In this fashion, thedistribution or uptake of the drug may be monitored by thepharmacodynamic marker. Similarly, the presence or quantity of thepharmacodynamic marker may be related to the presence or quantity of themetabolic product of a drug, such that the presence or quantity of themarker is indicative of the relative breakdown rate of the drug in vivo.Pharmacodynamic markers are of particular use in increasing thesensitivity of detection of drug effects, particularly when the drug isadministered in low doses. Since even a small amount of a drug may besufficient to activate multiple rounds of marker (e.g., an MTP-1, anOAT, an HST-1, a TP-2, a PLTR-1, a TFM-2, a TFM-3, a 67118, a 67067, a62092, a HAAT, an HST-4 and/or an HST-5 marker) transcription orexpression, the amplified marker may be in a quantity which is morereadily detectable than the drug itself. Also, the marker may be moreeasily detected due to the nature of the marker itself; for example,using the methods described herein, anti-MTP-1, anti-OAT, anti-HST-1,anti-TP-2, anti-PLTR-1, anti-TFM-2, anti-TFM-3, anti-67118, anti-67067,anti-62092, anti-HAAT, anti-HST-4 and/or anti-HST-5 antibodies may beemployed in an immune-based detection system for an MTP-1, an OAT, anHST-1, a TP-2, a PLTR-1, a TFM-2, a TFM-3, a 67118, a 67067, a 62092, aHAAT, an HST-4 and/or an HST-5 polypeptide marker, or MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4- and/orHST-5-specific radiolabeled probes may be used to detect an MTP-1, anOAT, an HST-1, a TP-2, a PLTR-1, a TFM-2, a TFM-3, a 67118, a 67067, a62092, a HAAT, an HST-4 and/or an HST-5 mRNA marker. Furthermore, theuse of a pharmacodynamic marker may offer mechanism-based prediction ofrisk due to drug treatment beyond the range of possible directobservations. Examples of the use of pharmacodynamic markers in the artinclude: Matsuda et al. U.S. Pat. No. 6,033,862; Hattis et al. (1991)Env. Health Perspect. 90: 229-238; Schentag (1999) Am. J. Health-Syst.Pharm. 56 Suppl. 3: S21-S24; and Nicolau (1999) Am, J. Health-Syst.Pharm. 56 Suppl. 3: S16-S20.

The MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or the HST-5 molecules of the invention are also usefulas pharmacogenomic markers. As used herein, a “pharmacogenomic marker”is an objective biochemical marker which correlates with a specificclinical drug response or susceptibility in a subject (see, e.g., McLeodet al. (1999) Eur. J. Cancer 35(12): 1650-1652). The presence orquantity of the pharmacogenomic marker is related to the predictedresponse of the subject to a specific drug or class of drugs prior toadministration of the drug. By assessing the presence or quantity of oneor more pharmacogenomic markers in a subject, a drug therapy which ismost appropriate for the subject, or which is predicted to have agreater degree of success, may be selected. For example, based on thepresence or quantity of RNA, or polypeptide (e.g., MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5 polypeptides or RNAs) for specific tumor markers in a subject, adrug or course of treatment may be selected that is optimized for thetreatment of the specific tumor likely to be present in the subject.Similarly, the presence or absence of a specific sequence mutation inMTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5 DNA may correlate MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 drugresponse. The use of pharmacogenomic markers therefore permits theapplication of the most appropriate treatment for each subject withouthaving to administer the therapy.

F. Electronic Apparatus-Readable Media and Arrays

Electronic apparatus readable media comprising MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5sequence information is also provided. As used herein, “MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 sequence information” refers to any nucleotide and/or aminoacid sequence information particular to the MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5molecules of the present invention, including but not limited tofull-length nucleotide and/or amino acid sequences, partial nucleotideand/or amino acid sequences, polymorphic sequences including singlenucleotide polymorphisms (SNPs), epitope sequences, and the like.Moreover, information “related to” said MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 sequenceinformation includes detection of the presence or absence of a sequence(e.g., detection of expression of a sequence, fragment, polymorphism,etc.), determination of the level of a sequence (e.g., detection of alevel of expression, for example, a quantitative detection), detectionof a reactivity to a sequence (e.g., detection of protein expressionand/or levels, for example, using a sequence-specific antibody), and thelike. As used herein, “electronic apparatus readable media” refers toany suitable medium for storing, holding or containing data orinformation that can be read and accessed directly by an electronicapparatus. Such media can include, but are not limited to: magneticstorage media, such as floppy discs, hard disc storage medium, andmagnetic tape; optical storage media such as compact disc; electronicstorage media such as RAM, ROM, EPROM, EEPROM and the like; general harddisks and hybrids of these categories such as magnetic/optical storagemedia. The medium is adapted or configured for having recorded thereon asequence of the present invention.

As used herein, the term “electronic apparatus” is intended to includeany suitable computing or processing apparatus or other deviceconfigured or adapted for storing data or information. Examples ofelectronic apparatus suitable for use with the present invention includestand-alone computing apparatus; networks, including a local areanetwork (LAN), a wide area network (WAN) Internet, Intranet, andExtranet; electronic appliances such as a personal digital assistants(PDAs), cellular phone, pager and the like; and local and distributedprocessing systems.

As used herein, “recorded” refers to a process for storing or encodinginformation on the electronic apparatus readable medium. Those skilledin the art can readily adopt any of the presently known methods forrecording information on known media to generate manufactures comprisingthe MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5 sequence information.

A variety of software programs and formats can be used to store thesequence information on the electronic apparatus readable medium. Forexample, the sequence information can be represented in a wordprocessing text file, formatted in commercially-available software suchas WordPerfect and Microsoft Word, or represented in the form of anASCII file, stored in a database application, such as DB2, Sybase,Oracle, or the like, as well as in other forms. Any number ofdataprocessor structuring formats (e.g., text file or database) may beemployed in order to obtain or create a medium having recorded thereonthe MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5 sequence information.

By providing MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5 sequence information in readableform, one can routinely access the sequence information for a variety ofpurposes. For example, one skilled in the art can use the sequenceinformation in readable form to compare a target sequence or targetstructural motif with the sequence information stored within the datastorage means. Search means are used to identify fragments or regions ofthe sequences of the invention which match a particular target sequenceor target motif.

The present invention therefore provides a medium for holdinginstructions for performing a method for determining whether a subjecthas a MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5-associated disease or disorder (e.g., ablood sugar or metabolic disorder) or a pre-disposition to a MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5-associated disease or disorder, wherein the methodcomprises the steps of determining MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 sequenceinformation associated with the subject and based on the MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5 sequence information, determining whether the subject has aMTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5-associated disease or disorder (e.g., a bloodsugar or metabolic disorder) or a pre-disposition to a MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5-associated disease or disorder and/or recommending aparticular treatment for the disease, disorder or pre-disease condition.

The present invention further provides in an electronic system and/or ina network, a method for determining whether a subject has a MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5-associated disease or disorder (e.g., a blood sugar ormetabolic disorder) or a pre-disposition to a disease associated with aMTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5 wherein the method comprises the steps ofdetermining MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 sequence information associated with thesubject, and based on the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5 sequence information,determining whether the subject has a MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5-associateddisease or disorder (e.g., a blood sugar or metabolic disorder) or apre-disposition to a MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5-associated disease ordisorder, and/or recommending a particular treatment for the disease,disorder or pre-disease condition. The method may further comprise thestep of receiving phenotypic information associated with the subjectand/or acquiring from a network phenotypic information associated withthe subject.

The present invention also provides in a network, a method fordetermining whether a subject has a MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5-associateddisease or disorder (e.g., a blood sugar or metabolic disorder) or apre-disposition to a MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5-associated disease ordisorder associated with MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5, said method comprisingthe steps of receiving MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5 sequence information fromthe subject and/or information related thereto, receiving phenotypicinformation associated with the subject, acquiring information from thenetwork corresponding to MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5 and/or a MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or corresponding to a HST-5-associated disease or disorder (e.g., ablood sugar or metabolic disorder), and based on one or more of thephenotypic information, the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 information (e.g.,sequence information and/or information related thereto), and theacquired information, determining whether the subject has a MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5-associated disease or disorder (e.g., a blood sugar ormetabolic disorder) or a pre-disposition to a MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5-associated disease or disorder. The method may further comprisethe step of recommending a particular treatment for the disease,disorder or pre-disease condition.

The present invention also provides a business method for determiningwhether a subject has a MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5-associated disease ordisorder (e.g., a blood sugar or metabolic disorder) or apre-disposition to a MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5-associated disease ordisorder, said method comprising the steps of receiving informationrelated to MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 (e.g., sequence information and/orinformation related thereto), receiving phenotypic informationassociated with the subject, acquiring information from the networkrelated to MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5 and/or related to a MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5-associated disease or disorder (e.g., a blood sugar or metabolicdisorder), and based on one or more of the phenotypic information, theMTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4 and/or HST-5 information, and the acquired information,determining whether the subject has a MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5-associateddisease or disorder (e.g., a blood sugar or metabolic disorder) or apre-disposition to a MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5-associated disease ordisorder. The method may further comprise the step of recommending aparticular treatment for the disease, disorder or pre-disease condition.

The invention also includes an array comprising a MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5 sequence of the present invention. The array can be used to assayexpression of one or more genes in the array. In one embodiment, thearray can be used to assay gene expression in a tissue to ascertaintissue specificity of genes in the array. In this manner, up to about7600 genes can be simultaneously assayed for expression, one of whichcan be MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4 and/or HST-5. This allows a profile to be developedshowing a battery of genes specifically expressed in one or moretissues.

In addition to such qualitative determination, the invention allows thequantitation of gene expression. Thus, not only tissue specificity, butalso the level of expression of a battery of genes in the tissue isascertainable. Thus, genes can be grouped on the basis of their tissueexpression per se and level of expression in that tissue. This isuseful, for example, in ascertaining the relationship of gene expressionbetween or among tissues. Thus, one tissue can be perturbed and theeffect on gene expression in a second tissue can be determined. In thiscontext, the effect of one cell type on another cell type in response toa biological stimulus can be determined. Such a determination is useful,for example, to know the effect of cell-cell interaction at the level ofgene expression. If an agent is administered therapeutically to treatone cell type but has an undesirable effect on another cell type, theinvention provides an assay to determine the molecular basis of theundesirable effect and thus provides the opportunity to co-administer acounteracting agent or otherwise treat the undesired effect. Similarly,even within a single cell type, undesirable biological effects can bedetermined at the molecular level. Thus, the effects of an agent onexpression of other than the target gene can be ascertained andcounteracted.

In another embodiment, the array can be used to monitor the time courseof expression of one or more genes in the array. This can occur invarious biological contexts, as disclosed herein, for exampledevelopment of a MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4 and/or HST-5-associated disease or disorder(e.g., a blood sugar or metabolic disorder), progression of MTP-1, OAT,HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4and/or HST-5-associated disease or disorder (e.g., a blood sugar ormetabolic disorder), and processes, such a cellular transformationassociated with the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3,67118, 67067, 62092, HAAT, HST-4 and/or HST-5-associated disease ordisorder.

The array is also useful for ascertaining the effect of the expressionof a gene on the expression of other genes in the same cell or indifferent cells (e.g., ascertaining the effect of MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/orHST-5 expression on the expression of other genes). This provides, forexample, for a selection of alternate molecular targets for therapeuticintervention if the ultimate or downstream target cannot be regulated.

The array is also useful for ascertaining differential expressionpatterns of one or more genes in normal and abnormal cells. Thisprovides a battery of genes (e.g., including MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5)that could serve as a molecular target for diagnosis or therapeuticintervention.

The contents of the Sequence Listing are submitted herewith on compactdisc in a Word file named “sequence listing.doc” and are incorporatedherein by this reference. The compact disc was created on Jan. 21, 2005,and sequence listing.doc has 620 kilobytes.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures and the Sequence Listing, areincorporated herein by reference.

EXAMPLES Example 1 Identification and Characterization of Human MTP-1cDNA

In this example, the identification and characterization of the geneencoding human MTP-1 (clone Fbh38594) is described.

Isolation of the MTP-1 cDNA

The invention is based, at least in part, on the discovery of a humangenes encoding a novel protein, referred to herein as MTP-1. The entiresequence of human clones Fbh38594, was determined and found to containan open reading frame termed human “MTP-1”. The MTP-1 protein sequenceset forth in SEQ ID NO:2 comprises about 2144 amino acids. The codingregion (open reading frame) of SEQ ID NO:1, is set forth as SEQ ID NO:3.

Analysis of the Human MTP-1 Molecule

An analysis of the possible cellular localization of the MTP-1 proteinbased on its amino acid sequence was performed using the methods andalgorithms described in Nakai and Kanehisa (1992) Genomics 14:897-911,and at http://psort.nibb.ac.jp. The results of the analysis show thathuman MTP-1 (SEQ ID NO:2) may be localized to the endoplasmic reticulum,vesicles of the secretory system, and the nucleus.

A search of the amino acid sequence of MTP-1 was performed against theMemsat database (FIG. 1). This search resulted in the identification oftwelve transmembrane domains in the amino acid sequence of human MTP-1(SEQ ID NO:2) at about residues 23-40, 548-564, 588-612, 624-646,653-675, 1006-1023, 1236-1258, 1534-1556, 1587-1603, 1645-1667,1732-1749, 1931-1947.

A search of the amino acid sequence of MTP-1 was also performed againstthe HMM database. This search resulted in the identification of two “ABCtransporter domains” in the amino acid sequence of MTP-1 (SEQ ID NO:2)at about residues 832-1012 and about 1818-1999 (scores: 206.0 and 144.2,respectively). Further domain motifs were identified by using the aminoacid sequence of MTP-1 (SEQ ID NO:2) to search through the ProDomdatabase (http://protein.toulouse.inra.fr/prodom.html). Numerous matchesagainst protein domains described as ATP-binding transporters, ABCtransporters, ABCR transporters, ABC-C transporters and the like wereidentified.

A search was also performed against the Prosite database, and resultedin the identification of two “ATP/GTP binding site motifs (P-loop)” atresidues 839-846, and 1825-1832 (Prosite accession number PS00017). Thissearch also revealed an “ABC transporter family signature motif” atresidues 938-952 (Prosite accession number PS00211).

BLASTN analysis using the nucleotide sequence of human MTP-1 resulted inthe identification of a partial cDNA having significant identity tonucleotides 2852-2987 of SEQ ID NO:1. This partial cDNA is described asbelonging to the ATP binding cassette (ABC) transporter protein family,etiologically involved in cholesterol driven atherogenic processes andinflammatory diseases like psoriasis, lupus erythematosus and others.

In combination with the other examples described herein, these datasuggest that MTP-1 is a novel ABC transporter molecule, involved inlipid metabolism and/or inflammation and/or hematopoiesis.

Example 2 Expression of Recombinant MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and HST-5 Polypeptides inBacterial Cells

In this example, In this example, human MTP-1, human OAT, human HST-1,human TP-2, human PLTR-1, human TFM-2, human TFM-3, human 67118, human67067, human 62092, human HAAT, human HST-4 and/or human HST-5 isexpressed as a recombinant glutathione-S-transferase (GST) fusionpolypeptide in E. coli and the fusion polypeptide is isolated andcharacterized. Specifically, MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4 and/or HST-5 is fused to GST andthis fusion polypeptide is expressed in E. coli, e.g., strain PEB199.Expression of the GST- MTP-1, GST-OAT, GST-HST-1, GST-TP-2, GST-PLTR-1,GST-TFM-2, GST-TFM-3, GST-67118, GST-67067, GST-62092, GST-HAAT,GST-HST-4, or GST-HST-5 fusion protein in PEB199 is induced with IPTG.The recombinant fusion polypeptide is purified from crude bacteriallysates of the induced PEB199 strain by affinity chromatography onglutathione beads. Using polyacrylamide gel electrophoretic analysis ofthe polypeptide purified from the bacterial lysates, the molecularweight of the resultant fusion polypeptide is determined.

Example 3 Expression of Recombinant MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4 and HST-5 Protein in CosCells

To express the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4, or HST-5 gene in COS cells, the pcDNA/Ampvector by Invitrogen Corporation (San Diego, Calif.) is used. Thisvector contains an SV40 origin of replication, an ampicillin resistancegene, an E. coli replication origin, a CMV promoter followed by apolylinker region, and an SV40 intron and polyadenylation site. A DNAfragment encoding the entire MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4, or HST-5 protein and an HA tag(Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its3′ end of the fragment is cloned into the polylinker region of thevector, thereby placing the expression of the recombinant protein underthe control of the CMV promoter.

To construct the plasmid, the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4, or HST-5 DNA sequence isamplified by PCR using two primers. The 5′ primer contains therestriction site of interest followed by approximately twentynucleotides of the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118,67067, 62092, HAAT, HST-4, or HST-5 coding sequence starting from theinitiation codon; the 3′ end sequence contains complementary sequencesto the other restriction site of interest, a translation stop codon, theHA tag or FLAG tag and the last 20 nucleotides of the MTP-1, OAT, HST-1,TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4, or HST-5coding sequence. The PCR amplified fragment and the pCDNA/Amp vector aredigested with the appropriate restriction enzymes and the vector isdephosphorylated using the CIAP enzyme (New England Biolabs, Beverly,Mass.). Preferably the two restriction sites chosen are different sothat the MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067,62092, HAAT, HST-4, or HST-5 gene is inserted in the correctorientation. The ligation mixture is transformed into E. coli cells(strains HB101, DH5α, SURE, available from Stratagene Cloning Systems,La Jolla, Calif., can be used), the transformed culture is plated onampicillin media plates, and resistant colonies are selected. PlasmidDNA is isolated from transformants and examined by restriction analysisfor the presence of the correct fragment.

COS cells are subsequently transfected with the MTP-1, OAT, HST-1, TP-2,PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4, orHST-5-pcDNA/Amp plasmid DNA using the calcium phosphate or calciumchloride co-precipitation methods, DEAE-dextran-mediated transfection,lipofection, or electroporation. Other suitable methods for transfectinghost cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989. The expression of the MTP-1 polypeptide is detected byradiolabeling (³⁵S-methionine or ³⁵S-cysteine available from NEN,Boston, Mass., can be used) and immunoprecipitation (Harlow, E. andLane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1988) using an HA specific monoclonalantibody. Briefly, the cells are labeled for 8 hours with ³⁵S-methionine(or ³⁵S-cysteine). The culture media are then collected and the cellsare lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1%SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culturemedia are precipitated with an HA-specific monoclonal antibody.Precipitated polypeptides are then analyzed by SDS-PAGE.

Alternatively, DNA containing the MTP-1, OAT, HST-1, TP-2, PLTR-1,TFM-2, TFM-3, 67118, 67067, 62092, HAAT, HST-4, or HST-5 coding sequenceis cloned directly into the polylinker of the pCDNA/Amp vector using theappropriate restriction sites. The resulting plasmid is transfected intoCOS cells in the manner described above, and the expression of theMTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2, TFM-3, 67118, 67067, 62092,HAAT, HST-4, or HST-5 polypeptide is detected by radiolabeling andimmunoprecipitation using an MTP-1, OAT, HST-1, TP-2, PLTR-1, TFM-2,TFM-3, 67118, 67067, 62092, HAAT, HST-4, or HST-5 specific monoclonalantibody.

Example 4 Tissue Distribution of MTP-1 mRNA

In this example, endogenous gene expression was determined using thePerkin-Elmer/AsI 7700 Sequence Detection System which employs TaqMantechnology. Briefly, TaqMan technology relies on standard RT-PCR withthe addition of a third gene-specific oligonucleotide (referred to as aprobe) which has a fluorescent dye coupled to its 5′ end (typically6-FAM) and a quenching dye at the 3′ end (typically TAMRA). When thefluorescently tagged oligonucleotide is intact, the fluorescent signalfrom the 5′ dye is quenched. As PCR proceeds, the 5′ to 3′ nucleolyticactivity of taq polymerase digests the labeled primer, producing a freenucleotide labeled with 6-FAM, which is now detected as a fluorescentsignal. The PCR cycle where fluorescence is first released and detectedis directly proportional to the starting amount of the gene of interestin the test sample, thus providing a way of quantitating the initialtemplate concentration. Samples can be internally controlled by theaddition of a second set of primers/probe specific for a housekeepinggene such as GAPDH which has been labeled with a different fluor on the5′ end (typically JOE).

To determine the level of MTP-1 in various tissues a primer/probe setwas designed using Primer Express software and primary cDNA sequenceinformation. Total RNA was prepared from a series of tissues using anRNeasy kit from Qiagen First strand cDNA was prepared from one μg totalRNA using an oligo dT primer and Superscript II reverse transcriptase(GIBCO-BRL). cDNA obtained from approximately 50 ng total RNA was usedper TaqMan reaction. An array of human tissues were tested. The resultsof one such analysis are depicted in FIGS. 2A-C. Expression was greatestin brain, vein, adipose, skin, fetal liver, tonsil, and lymph node.Expression was also noted in liver, colon, skeletal muscle, kidney,lung, thyroid, bone marrow, testis, placenta, fetal heart, spleen, andthymus.

In addition, a second array of human tissues was tested according to theabove-described Taqman procedure, the array including additional samplesof the erythroid and hematopoietic lineage. Notably, in addition toincreased expression in tonsil and lymph node tissue, significantexpression was also observed in bone marrow mononuclear cells,megakaryocytes and neutrophils, with quite dramatic expression beingdetected in erythroid cells. TABLE I Tissue Type Mean β 2 Mean ∂∂ CtExpression Artery normal 34.5 23.43 9.08 1.8478 Aorta diseased 33.9523.5 8.46 2.8398 Vein normal 37.27 21.48 13.8 0 Coronary SMC 34.41 22.0710.36 0.7635 HUVEC 30.69 22.52 6.18 13.7922 Hemangioma 33.1 21.05 10.070.9335 Heart normal 33.34 22.2 9.14 1.7664 Heart CHF 33.51 22.09 9.431.4548 Kidney 30.5 21.41 7.11 7.2641 Skeletal Muscle 31.26 23.18 6.0914.68 Adipose normal 34.95 21.89 11.07 0.4652 Pancreas 32.16 23.35 6.838.82 primary osteoblasts 30.86 21.93 6.93 8.1725 Osteoclasts 32.65 18.9311.72 0.2964 Skin normal 33.91 23.2 8.72 2.3633 Spinal cord normal 33.0122.13 8.89 2.1006 Brain Cortex normal 30.45 23.5 4.96 32.1286 BrainHypothalamus normal 33.28 23.63 7.66 4.9444 Nerve 35.15 23.34 9.82 0 DRG(Dorsal Root Ganglion) 30.59 22.94 5.66 19.8461 Breast normal 33.9422.18 9.77 1.1493 Breast tumor 32.81 21.99 8.83 2.1974 Ovary normal34.65 21.23 11.43 0.3624 Ovary Tumor 30.8 19.73 9.09 1.8414 ProstateNormal 32.9 20.93 9.98 0.9868 Prostate Tumor 32.58 21.3 9.29 1.5919Salivary glands 32.64 20.8 9.85 1.0836 Colon normal 34.74 19.81 12.950.1268 Colon Tumor 31.76 22.48 7.29 6.3899 Lung normal 31.25 19.34 9.911.0358 Lung tumor 29.82 21.58 6.25 13.0935 Lung 32.22 19.77 10.47 0.7075Colon 32.47 18.8 11.69 0.3037 Liver normal 36.3 21.14 13.17 0 Liverfibrosis 33.8 21.87 9.94 1.0216 Spleen normal 30.26 19.77 8.5 2.7621Tonsil normal 28.23 19.76 6.48 11.2028 Lymph node normal 29.54 21.326.24 13.2763 Small intestine normal 35.99 21.38 12.63 0 Macrophages33.92 18.14 13.8 0.0701 Synovium 34.2 20.9 11.32 0.3925 BM-MNC 28.8820.05 6.84 8.6986 Activated PBMC 32.9 19.48 11.43 0.3624 Neutrophils28.03 19.42 6.62 10.1667 Megakaryocytes 27.09 20.12 4.97 31.7962Erythroid 25.68 21.81 1.88 271.6837 positive control 30.11 21.34 6.779.1628

To further investigate the high expression in hematopoietic tissue,MTP-1 expression levels were measured in various hematopoietic cells byquantitative PCR using the Taqman™ procedure as described above. Therelative levels of MTP-1 expression in various hematopoietic andnon-hemapoietic cells is depicted in Table II. TABLE II Expression onMTP-1 in various types of hematopoietic cells. Fam Mean Relative 38594Vic Mean Beta2 Expression Lung MPI 131 29 19 18 Kidney MPI 58 28 21 255Brain MPI 167 33 24 34 Heart PIT 273 34 20 2 Colon MPI 60 32 21 10 NHLFCTN 49 hr 30 19 9 NHLF TGF 10 ng 30 19 12 hepG2 CTN 29 20 67 Tonsil MPI37 26 19 204 Lymph nodes NDR 79 26 19 225 spleen MPI 380 23 17 287 Fetalliver MPI 133 30 21 65 pooled liver 31 20 16 Liv Fib NDR 190 36 25 16Liv Fib NDR 191 30 20 37 Liv Fib NDR 194 35 25 38 Liv Fib NDR 113 31 197 Th1 48 hr M4 30 17 3 Th1 48 hr M5 30 17 3 Th2 48 hr M5 30 17 3 Grans27 20 218 CD19 28 18 18 CD14 30 17 2 PBMC mock 25 16 63 PBMC PHA 27 16 8PBMC IL 10 28 17 8 NHBE mock 32 20 8 NHBE IL13-1 32 21 10 BM-MNC 32 2110 mPB CD34+ 27 20 351 ABM CD34+ 29 19 18 Erythroid 30 20 22 Megs 31 184 Neutrophil 30 19 14 mBM CD11b+ 33 18 1 mBM CD15+ 32 18 2 mBM CD11b− 3018 4 BM/GPA 28 20 91 BM CD71 27 18 60 HepG2 A 29 22 202 HepG2 2.12-a 2822 412 NTC 40 40

Notably, MTP-1 expression was increased in non-hemapoietic cells such asHepG2, brain, liver and kidney. Interesting, expression was mostincreased in hematopoietic cells such as CD34-positive murine peripheralblood cells. Expression was also significantly increased in otherhemapoietic cells such as glycophorin A-positive bone marrow cells(“BM-GPA”), CD71-positive bone marrow cells (BM-CD71”), mock-treatedperipheral blood mononuclear cells, granulocytes, tonsils, lymph nodesand spleen. These data indicate that MTP-1 is a novel ABC-transportermolecule that is preferentially expressed in various hemapoietic cells.

Example 5 Identification and Characterization of Human Oat cDNA

In this example, the identification and characterization of the genesencoding human OAT4 (clone Fbh57312) and human OAT5 (clone Fbh53659) isdescribed.

Isolation of the Human OAT cDNA

The invention is based, at least in part, on the discovery of genesencoding novel members of the organic anion transporter family. Theentire sequence of human clone Fbh57312 was determined and found tocontain an open reading frame termed human “OAT4”. The entire sequenceof human clone Fbh53659 was determined and found to contain an openreading frame termed human “OAT5”.

The nucleotide sequence encoding the human OAT4 is shown is set forth asSEQ ID NO:4. The protein encoded by this nucleic acid comprises about550 amino acids and has the amino acid sequence set forth as SEQ IDNO:5. The coding region (open reading frame) of SEQ ID NO:4 is set forthas SEQ ID NO:6.

The nucleotide sequence encoding the human OAT5 is set forth as SEQ IDNO:7. The protein encoded by this nucleic acid comprises about 724 aminoacids and has the amino acid sequence set forth as SEQ ID NO:8. Thecoding region (open reading frame) is set forth as SEQ ID NO:9.

Analysis of the Human OAT Molecules

The amino acid sequences of human OAT4 and OAT5 were analyzed using theprogram PSORT (available online) to predict the localization of theproteins within the cell. This program assesses the presence ofdifferent targeting and localization amino acid sequences within thequery sequence. The results of the analyses show that human OAT4 may belocalized to the endoplasmic reticulum, the nucleus, or themitochondria. The results of the analyses further show that human OAT 5may be localized to the endoplasmic reticulum, vacuoles, secretoryvesicles, or the mitochondria.

Additionally, searches of the amino acid sequences of human OAT4 andOAT5 were performed against the Memsat database. These searches resultedin the identification of 12 transmembrane domains in the amino acidsequence of human OAT4 at residues 1-31, 148-165, 172-195, 202-219,228-252, 260-276, 347-365, 375-399, 406-422, 431-451, 466-484, and495-512 of SEQ ID NO:5 (FIG. 4). These searches further resulted in theidentification of 12 transmembrane domains in the amino acid sequence ofhuman OAT5 at residues 106-130, 143-166, 174-191, 230-254, 265-284,314-335, 382-405, 419-443, 456-473, 579-603, 613-636, and 667-690 of SEQID NO:8 (FIG. 5).

Searches of the amino acid sequences of human OAT4 and OAT5 were alsoperformed against the HMM database. These searches resulted in theidentification of a “sugar (and other) transporter domain” at aboutresidues 103-527 (score=34.7) of SEQ ID NO:5 . These searches furtherresulted in the identification of a “sugar (and other) transporterdomain” at about residues 141-555 of SEQ ID NO:8.

Searches of the amino acid sequence of human OAT were further performedagainst the Prosite database. These searches resulted in theidentification of two ATP/GTP-binding site motif A (P-loop) domains inthe amino acid sequence of human OAT5 at about residues 343-350 and360-367 of SEQ ID NO:8. These searches also resulted in theidentification of a number of potential N-glycosylation sites, proteinkinase C phosphorylation sites, casein kinase II phosphorylation sites,N-myristoylation sites, amidation sites, and leucine zipper patterns inthe amino acid sequence of human OAT4. These searches further resultedin the identification in the amino acid sequence of human OAT5 of apotential cAMP- and cGMP-dependent protein kinase phosphorylation siteand an number of potential N-glycosylation sites, protein kinase Cphosphorylation sites, casein kinase II phosphorylation sites, andN-myristoylation sites.

Tissue Distribution of OAT mRNA

This example describes the tissue distribution of human OAT mRNA, as maybe determined using in situ hybridization analysis. For in situanalysis, various tissues, e.g., tissues obtained from brain, are firstfrozen on dry ice. Ten-micrometer-thick sections of the tissues arepostfixed with 4% formaldehyde in DEPC-treated 1× phosphate-bufferedsaline at room temperature for 10 minutes before being rinsed twice inDEPC 1× phosphate-buffered saline and once in 0.1 M triethanolamine-HCl(pH 8.0). Following incubation in 0.25% acetic anhydride-0.1 Mtriethanolamine-HCl for 10 minutes, sections are rinsed in DEPC 2×SSC(1×SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Tissue is thendehydrated through a series of ethanol washes, incubated in 100%chloroform for 5 minutes, and then rinsed in 100% ethanol for 1 minuteand 95% ethanol for 1 minute and allowed to air dry.

Hybridizations are performed with ³⁵S-radiolabeled (5×10⁷ cpm/ml) cRNAprobes. Probes are incubated in the presence of a solution containing600 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 0.01% sheared salmon spermDNA, 0.01% yeast tRNA, 0.05% yeast total RNA type X1, 1× Denhardt'ssolution, 50% formamide, 10% dextran sulfate, 100 mM dithiothreitol,0.1% sodium dodecyl sulfate (SDS), and 0.1% sodium thiosulfate for 18hours at 55° C.

After hybridization, slides are washed with 2×SSC. Sections are thensequentially incubated at 37° C. in TNE (a solution containing 10 mMTris-HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA), for 10 minutes, in TNEwith 10 μg of RNase A per ml for 30 minutes, and finally in TNE for 10minutes. Slides are then rinsed with 2×SSC at room temperature, washedwith 2×SSC at 50° C. for 1 hour, washed with 0.2×SSC at 55° C. for 1hour, and 0.2×SSC at 60° C. for 1 hour. Sections are then dehydratedrapidly through serial ethanol-0.3 M sodium acetate concentrationsbefore being air dried and exposed to Kodak Biomax MR scientific imagingfilm for 24 hours and subsequently dipped in NB-2 photoemulsion andexposed at 4° C. for 7 days before being developed and counter stained.

Analysis of Human OAT Expression Using the Taqman Procedure

The Taqman™ procedure is a quantitative, real-time PCR-based approach todetecting mRNA. The RT-PCR reaction exploits the 5′ nuclease activity ofAmpliTaq Gold™ DNA Polymerase to cleave a TaqMan™ probe during PCR.Briefly, cDNA was generated from the samples of interest and served asthe starting material for PCR amplification. In addition to the 5′ and3′ gene-specific primers, a gene-specific oligonucleotide probe(complementary to the region being amplified) was included in thereaction (i.e., the Taqman™ probe). The TaqMan™ probe included anoligonucleotide with a fluorescent reporter dye covalently linked to the5′ end of the probe (such as FAM (6-carboxyfluorescein), TET(6-carboxy-4,7,2′,7′-tetrachlorofluorescein), JOE(6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein), or VIC) and aquencher dye (TAMRA (6-carboxy-N,N,N′,N′-tetramethylrhodamine) at the 3′end of the probe.

During the PCR reaction, cleavage of the probe separated the reporterdye and the quencher dye, resulting in increased fluorescence of thereporter. Accumulation of PCR products was detected directly bymonitoring the increase in fluorescence of the reporter dye. When theprobe was intact, the proximity of the reporter dye to the quencher dyeresulted in suppression of the reporter fluorescence. During PCR, if thetarget of interest was present, the probe specifically annealed betweenthe forward and reverse primer sites. The 5′-3′ nucleolytic activity ofthe AmpliTaq™ Gold DNA Polymerase cleaved the probe between the reporterand the quencher only if the probe hybridized to the target. The probefragments were then displaced from the target, and polymerization of thestrand continued. The 3′ end of the probe was blocked to preventextension of the probe during PCR. This process occurred in every cycleand did not interfere with the exponential accumulation of product. RNAwas prepared using the trizol method and treated with DNase to removecontaminating genomic DNA. cDNA was synthesized using standardtechniques. Mock cDNA synthesis in the absence of reverse transcriptaseresulted in samples with no detectable PCR amplification of the controlGAPDH or β-actin gene confirming efficient removal of genomic DNAcontamination.

Taqman analysis showed that human OAT5 was highly expressed in thekidney, primary osteoblasts, brain cortex, lung, liver, bone marrowmononuclear cells (BM-MNC), and neutrophils (see FIG. 6).

Example 6 Identification and Characterization of Human HST-1 cDNA

In this example, the identification and characterization of the geneencoding human HST-1 (clone 57250) is described.

Isolation of the Human HST-1 cDNA

The invention is based, at least in part, on the discovery of a humangene encoding a novel polypeptide, referred to herein as human HST-1.The entire sequence of the human clone 57250 was determined and found tocontain an open reading frame termed human “HST-1.” The nucleotidesequence of the human HST-1 gene is set forth in the Sequence Listing asSEQ ID NO:12. The amino acid sequence of the human HST-1 expressionproduct is set forth in the Sequence Listing as SEQ ID NO:13. The HST-1polypeptide comprises 572 amino acids. The coding region (open readingframe) of SEQ ID NO:12 is set forth as SEQ ID NO:14.

Analysis of the Human HST-1 Molecules

The human HST-1 amino acid sequence was aligned with the amino acidsequence of the potent brain type organic ion transporter from Homosapiens (Accession No. AB040056) using the CLUSTAL W (1.74) multiplesequence alignment program. The results of the alignment are set forthin FIG. 9.

A search using the polypeptide sequence of SEQ ID NO:13 was performedagainst the HMM database in PFAM resulting in the identification of asugar transporter family domain in the amino acid sequence of humanHST-1 at about residues 117-536 of SEQ ID NO:13, a potential UL25 domainin the amino acid sequence of human HST-1 at about residues 577-597 ofSEQ ID NO:13 (score=3.0), and a potential sodium: galactoside symporterfamily domain in the amino acid sequence of human HST-1 at aboutresidues 287-541 of SEQ ID NO:13.

The amino acid sequence of human HST-1 was analyzed using the programPSORT (see the PSORT website) to predict the localization of theproteins within the cell. This program assesses the presence ofdifferent targeting and localization amino acid sequences within thequery sequence. The results of this analysis indicated that human HST-1may be localized to the endoplasmic reticulum, nucleus, secretoryvesicles or mitochondria.

Searches of the amino acid sequence of human HST-1 were furtherperformed against the Prosite database. These searches resulted in theidentification in the amino acid sequence of human HST-1 of a potentialN-glycosylation site, a number of potential protein kinase Cphosphorylation sites, a number of potential casein kinase IIphosphorylation sites, a number of potential N-myristoylation sites, anumber of potential amidation sites, a potential prokaryotic membranelipoprotein lipid attachment site, and a number of potential leucinezipper motifs.

A MEMSAT analysis of the polypeptide sequence of SEQ ID NO:13 was alsoperformed (FIG. 8), predicting twelve transmembrane domains in the aminoacid sequence of human HST-1 (SEQ ID NO:13) at about residues 20-36,150-167, 174-196, 204-220, 231-255, 263-282, 355-372, 387-405, 413-431,438-462, 469-485, and 505-521.

Further domain motifs were identified by using the amino acid sequenceof HST-1 (SEQ ID NO:13) to search through the ProDom database. Numerousmatches against protein domains described as “transporter organic cationMBOCT potent brain type”, “transporter organic cation aniontransmembrane glycoprotein monoamine”, “DNA packaging” and the like wereidentified.

Example 7 Tissue Distribution of Human HST-1 mRNA Using Taqman™ Analysis

This example describes the tissue distribution of human HST-1 mRNA in avariety of cells and tissues, as determined using the TaqMan™ procedure.The Taqman™ procedure is a quantitative, reverse transcription PCR-basedapproach for detecting mRNA. The RT-PCR reaction exploits the 5′nuclease activity of AmpliTaq Gold™ DNA Polymerase to cleave a TaqMan™probe during PCR. Briefly, cDNA was generated from the samples ofinterest, e.g., various human tissue samples, and used as the startingmaterial for PCR amplification. In addition to the 5′ and 3′gene-specific primers, a gene-specific oligonucleotide probe(complementary to the region being amplified) was included in thereaction (i.e., the Taqman™ probe). The TaqMan™ probe includes theoligonucleotide with a fluorescent reporter dye covalently linked to the5′ end of the probe (such as FAM (6-carboxyfluorescein), TET(6-carboxy-4,7,2′,7′-tetrachlorofluorescein), JOE(6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein), or VIC) and aquencher dye (TAMRA (6-carboxy-N,N,N′,N′-tetramethylrhodamine) at the 3′end of the probe.

During the PCR reaction, cleavage of the probe separates the reporterdye and the quencher dye, resulting in increased fluorescence of thereporter. Accumulation of PCR products is detected directly bymonitoring the increase in fluorescence of the reporter dye. When theprobe is intact, the proximity of the reporter dye to the quencher dyeresults in suppression of the reporter fluorescence. During PCR, if thetarget of interest is present, the probe specifically anneals betweenthe forward and reverse primer sites. The 5′-3′ nucleolytic activity ofthe AmpliTaq™ Gold DNA Polymerase cleaves the probe between the reporterand the quencher only if the probe hybridizes to the target. The probefragments are then displaced from the target, and polymerization of thestrand continues. The 3′ end of the probe is blocked to preventextension of the probe during PCR. This process occurs in every cycleand does not interfere with the exponential accumulation of product. RNAwas prepared using the trizol method and treated with DNase to removecontaminating genomic DNA. cDNA was synthesized using standardtechniques. Mock cDNA synthesis in the absence of reverse transcriptaseresulted in samples with no detectable PCR amplification of the controlgene confirms efficient removal of genomic DNA contamination.

As indicated in FIG. 10, strong expression of HST-1 was detected inhuman coronary smooth muscle cells and neutrophils, as well as in normalhuman pancreatic tissue and human lung tissue derived from normal,tumor, and chronic obstructive pulmonary disease samples. In addition,HST-1 expression was detected at moderate levels in normal ovary andlymph node tissues, breast tumor tissue, prostate tumor tissue, and inbone marrow mononuclear cells.

Example 8 Identification and Characterization of Human TP-2 cDNA

In this example, the identification and characterization of the geneencoding human TP-2 (clone 63760) is described.

Isolation of the Human TP-2 cDNA

The invention is based, at least in part, on the discovery of a humangene encoding a novel polypeptide, referred to herein as human TP-2. Theentire sequence of the human clone 63760 was determined and found tocontain an open reading frame termed human “TP-2.” The nucleotidesequence of the human TP-2 gene is set forth in the Sequence Listing asSEQ ID NO:15. The amino acid sequence of the human TP-2 expressionproduct is set forth in the Sequence Listing as SEQ ID NO:16. The TP-2polypeptide comprises 474 amino acids. The coding region (open readingframe) of SEQ ID NO:15 is set forth as SEQ ID NO:17.

Analysis of the Human TP-2 Molecules

The human TP-2 amino acid sequence was aligned with the amino acidsequence of the tetracycline-6-hydroxylase/oxygenase homolog gene fromSalmonella typhi (SEQ ID NO:18) using the CLUSTAL W (1.74) multiplesequence alignment program. The results of the alignment are set forthin FIG. 13.

A search using the polypeptide sequence of SEQ ID NO:16 was performedagainst the HMM database in PFAM resulting in the identification of apotential sugar transporter domain in the amino acid sequence of humanTP-2 at about residues 37-454 of SEQ ID NO:16 (score=−101.1), apotential LacY proton/sugar symporter domain in the amino acid sequenceof human TP-2 at about residues 39-383 of SEQ ID NO:16 (score=−336.7), apotential glutamine amidotransferases class-II domain in the amino acidsequence of human TP-2 at about residues 165-170 of SEQ ID NO:16(score=1.2), and a potential MCT domain in the amino acid sequence ofhuman TP-2 at about residues 33-458 of SEQ ID NO:16 (score=−167.8).

The amino acid sequence of human TP-2 was analyzed using the programPSORT (http://www.psort.nibb.ac.jp) to predict the localization of theproteins within the cell. This program assesses the presence ofdifferent targeting and localization amino acid sequences within thequery sequence. The results of this analysis show that human TP-2 may belocalized to the endoplasmic reticulum, secretory vesicles, ormitochondria.

Searches of the amino acid sequence of human TP-2 were further performedagainst the Prosite database. These searches resulted in theidentification in the amino acid sequence of human TP-2 of two potentialglycosaminoglycan attachment sites at about residues 176-179 and 464-467of SEQ ID NO:16, two potential cAMP- and cGMP-dependent protein kinasephosphorylation sites at about residues 108-111 and 460-463 of SEQ IDNO:16, a number of potential protein kinase C phosphorylation sites atabout residues 228-230, 253-255, and 260-262 of SEQ ID NO:16, a numberof potential casein kinase II phosphorylation sites at about residues28-31, 191-194, 247-250, and 463-466 of SEQ ID NO:16, a number ofpotential N-myristoylation sites at about residues 38-43, 75-80, 82-87,127-132, 187-192, 332-337, 403-408, 409-414, 415-420, and 445-450 of SEQID NO:16, one potential amidation site at about residues 106-109 of SEQID NO:16, and a potential prokaryotic membrane lipoprotein lipidattachment site at about residues 99-114 of SEQ ID NO:16.

A MEMSAT analysis of the polypeptide sequence of SEQ ID NO:16 was alsoperformed (FIG. 12), predicting eleven potential transmembrane domainsin the amino acid sequence of human TR-2 (SEQ ID NO:16) at aboutresidues 45-69, 80-102, 112-136, 167-190, 197-218, 288-310, 323-343,352-368, 375-391, 409-433, and 422-458. However, a structural,hydrophobicity, and antigenicity analysis (FIG. 11) resulted in theidentification of twelve transmembrane domains. Accordingly, the TP-2protein of SEQ ID NO:16 is predicted to have at least 12 transmembranedomains, which are identified in FIGS. 11 and 12 as transmembrane (TM)domains 1 through 12. TM1 is at about residues 45-69, TM2 is at aboutresidues 80-102, TM3 is at about residues 112-126, TM4 is at aboutresidues 133-156, TM5 is at about residues 167-190, TM6 is at aboutresidues 197-218, TM7 is at about residues 288-310, TM8 is at aboutresidues 323-343, TM9 is at about residues 352-368, TM10 is at aboutresidues 375-391, TM11 is at about residues 409-433, and TM12 is atabout residues 442-458.

A search of the amino acid sequence of human TP-2 was also performedagainst the ProDom database. These searches resulted in theidentification of a “kinase activity integral membrane domain” at aboutamino acid residues 36-235, a “transport integral membrane” at aboutamino acid residues 41-190, a “YFKF transporter MFS transmembranedomain” at about amino acid residues 45-229, a “multidrug transmembranedomain” at about amino acid residues 130-250, a “transporter-likepolyspecific organic subtransferable suppressing membrane tumor domain”at about amino acid residues 133-214, a “transport membrane domain” atabout amino acid residues 163-244, a “NORA domain” at about amino acidresidues 190-462, and a “family C2-domain” at about amino acid residues399-466 in the amino acid protein sequence of TP-2 (SEQ ID NO:16).

Example 9 Identification and Characterization of Human PLTR-1 cDNA

In this example, the identification and characterization of the geneencoding human PLTR-1 (clone 49938) is described.

Isolation of the Human PLTR-1 cDNA

The invention is based, at least in part, on the discovery of genesencoding novel members of the phospholipid transporter family. Theentire sequence of human clone Fbh49938 was determined and found tocontain an open reading frame termed human “PLTR-1”.

The nucleotide sequence encoding the human PLTR-1 is set forth as SEQ IDNO:19. The protein encoded by this nucleic acid comprises about 1190amino acids and has the amino acid sequence is set forth as SEQ IDNO:20. The coding region (open reading frame) of SEQ ID NO:19 is setforth as SEQ ID NO:21.

Analysis of the Human PLTR-1 Molecules

The amino acid sequence of human PLTR-1 was analyzed for the presence ofsequence motifs specific for P-type ATPases (as defined in Tang, X. etal. (1996) Science 272:1495-1497 and Fagan, M. J. and Saier, M. H.(1994) J. Mol. Evol. 38:57). These analyses resulted in theidentification of a P-type ATPase sequence 1 motif in the amino acidsequence of human PLTR-1 at residues 164-172 of SEQ ID NO:20. Theseanalyses also resulted in the identification of a P-type ATPase sequence2 motif in the amino acid sequence of human PLTR-1 at residues 389-398of SEQ ID NO:20. These analyses further resulted in the identificationof a P-type ATPase sequence 3 motif in the amino acid sequence of humanPLTR-1 at residues 812-822 of SEQ ID NO:20.

The amino acid sequence of human PLTR-1 was also analyzed for thepresence of phospholipid transporter specific amino acid residues (asdefined in Tang, X. et al. (1996) Science 272:1495-1497). These analysesresulted in the identification of phospholipid transporter specificamino acid residues in the amino acid sequence of human PLTR-1 at aboutresidues 164, 165, 168, 390, 812, 821, and 822 of SEQ ID NO:20 (FIGS.14A-B).

The amino acid sequence of human PLTR-1 was also analyzed for thepresence of large extramembrane domains. An N-terminal largeextramembrane domain was identified in the amino acid sequence of humanPLTR-1 at residues 95-275 of SEQ ID NO:20. A C-terminal largeextramembrane domain was identified in the amino acid sequence of humanPLTR-1 at residues 345-879 of SEQ ID NO:20.

The amino acid sequence of human PLTR-1 was further analyzed using theprogram PSORT (available online; see Nakai, K. and Kanehisa, M. (1992)Genomics 14:897-911) to predict the localization of the proteins withinthe cell. This program assesses the presence of different targeting andlocalization amino acid sequences within the query sequence. The resultsof the analyses show that human PLTR-1 is most likely localized to theendoplasmic reticulum or to vesicles of the secretory system.

Analysis of the amino acid sequence of human PLTR-1 was performed usingMEMSAT. This analysis resulted in the identification of 10 possibletransmembrane domains in the amino acid sequence of human PLTR-1 atabout residues 55-71, 78-94, 276-298, 320-344, 880-897, 904-924,954-977, 993-1011, 1022-1038, and 1066-1084 of SEQ ID NO:20 (see FIGS.14A-B and 15).

Searches of the amino acid sequence of human PLTR-1 were furtherperformed against the Prosite database. These searches resulted in theidentification of an “E1-E2 ATPases phosphorylation site” at aboutresidues 498-504 of SEQ ID NO:20 (see FIGS. 14A-B). These searches alsoresulted in the identification in the amino acid sequence of humanPLTR-1 of a potential N-glycosylation site (at about amino acid residues579-582) and a number of potential cAMP- and cGMP-dependent proteinkinase phosphorylation sites (at about residues 265-268, 367-370,542-545, and 1171-1174), protein kinase C phosphorylation sites (atabout residues 36-38, 259-261, 391-393, 514-516, 687-689, 723-725,739-741, 1098-1100, 1124-1126, 1143-1145, 1158-1160, and 1168-1170),casein kinase II phosphorylation sites (at about residues 153-156,267-270, 370-373, 378-381, 413-416, 452-455, 493-496, 519-522, 573-576,580-583, 624-627, 631-634, 646-649, 705-708, 732-735, 744-747, 832-835,899-902, 980-983, 1132-1135, and 1164-1167), tyrosine phosphorylationsites (at about residues 17-23, 482-489, and 601-608), andN-myristoylation sites (at about residues 288-293, 497-502, 524-529,655-660, 728-733, 828-833, 961-966, 984-989, 1010-1015, 1055-1060, and1123-1128) in the amino acid sequence of SEQ ID NO:20.

A search of the amino acid sequence of human PLTR-1 was also performedagainst the ProDom database (available online through the CentreNational de la Recherche Scientifique, France; see Corpet, F. et al.(2000) Nucleic Acids Res. 28:267-269). This search resulted in theidentification of homology between the PLTR-1 protein and phospholipidtransporting ATPases (ProDom Accession Numbers PD004932, PD004982,PD030421, PD004657, PD304524, and PD116286).

Tissue Distribution of PLTR-1 mRNA Using in Situ Analysis

This example describes the tissue distribution of human PLTR-1 mRNA, asmay be determined using in situ hybridization analysis. For in situanalysis, various tissues, e.g., tissues obtained from brain or vessels,are first frozen on dry ice. Ten-micrometer-thick sections of thetissues are postfixed with 4% formaldehyde in DEPC-treated 1×phosphate-buffered saline at room temperature for 10 minutes beforebeing rinsed twice in DEPC 1× phosphate-buffered saline and once in 0.1M triethanolamine-HCl (pH 8.0). Following incubation in 0.25% aceticanhydride-0.1 M triethanolamine-HCl for 10 minutes, sections are rinsedin DEPC 2×SSC (1×SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Tissueis then dehydrated through a series of ethanol washes, incubated in 100%chloroform for 5 minutes, and then rinsed in 100% ethanol for 1 minuteand 95% ethanol for 1 minute and allowed to air dry.

Hybridizations are performed with ³⁵S-radiolabeled (5×10⁷ cpm/ml) cRNAprobes. Probes are incubated in the presence of a solution containing600 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 0.01% sheared salmon spermDNA, 0.01% yeast tRNA, 0.05% yeast total RNA type X1, 1× Denhardt'ssolution, 50% formamide, 10% dextran sulfate, 100 mM dithiothreitol,0.1% sodium dodecyl sulfate (SDS), and 0.1% sodium thiosulfate for 18hours at 55° C.

After hybridization, slides are washed with 2×SSC. Sections are thensequentially incubated at 37° C. in TNE (a solution containing 10 mMTris-HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA), for 10 minutes, in TNEwith 10 μg of RNase A per ml for 30 minutes, and finally in TNE for 10minutes. Slides are then rinsed with 2×SSC at room temperature, washedwith 2×SSC at 50° C. for 1 hour, washed with 0.2×SSC at 55° C. for 1hour, and 0.2×SSC at 60° C. for 1 hour. Sections are then dehydratedrapidly through serial ethanol-0.3 M sodium acetate concentrationsbefore being air dried and exposed to Kodak Biomax MR scientific imagingfilm for 24 hours and subsequently dipped in NB-2 photoemulsion andexposed at 4° C. for 7 days before being developed and counter stained.

Example 10 Analysis of Human PLTR-1 Expression

This example describes the expression of human PLTR-1 mRNA in varioushuman vessels, as determined using the TaqMan™ procedure.

The Taqman™ procedure is a quantitative, real-time PCR-based approach todetecting mRNA. The RT-PCR reaction exploits the 5′ nuclease activity ofAmpliTaq Gold™ DNA Polymerase to cleave a TaqMan™ probe during PCR.Briefly, cDNA was generated from the samples of interest and served asthe starting material for PCR amplification. In addition to the 5′ and3′ gene-specific primers, a gene-specific oligonucleotide probe(complementary to the region being amplified) was included in thereaction (i.e., the Taqman™ probe). The TaqMan™ probe included anoligonucleotide with a fluorescent reporter dye covalently linked to the5′ end of the probe (such as FAM (6-carboxyfluorescein), TET(6-carboxy-4,7,2′,7′-tetrachlorofluorescein), JOE(6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein), or VIC) and aquencher dye (TAMRA (6-carboxy-N,N,N′,N′-tetramethylrhodamine) at the 3′end of the probe.

During the PCR reaction, cleavage of the probe separated the reporterdye and the quencher dye, resulting in increased fluorescence of thereporter. Accumulation of PCR products was detected directly bymonitoring the increase in fluorescence of the reporter dye. When theprobe was intact, the proximity of the reporter dye to the quencher dyeresulted in suppression of the reporter fluorescence. During PCR, if thetarget of interest was present, the probe specifically annealed betweenthe forward and reverse primer sites. The 5′-3′ nucleolytic activity ofthe AmpliTaq™ Gold DNA Polymerase cleaved the probe between the reporterand the quencher only if the probe hybridized to the target. The probefragments were then displaced from the target, and polymerization of thestrand continued. The 3′ end of the probe was blocked to preventextension of the probe during PCR. This process occurred in every cycleand did not interfere with the exponential accumulation of product. RNAwas prepared using the trizol method and treated with DNase to removecontaminating genomic DNA. cDNA was synthesized using standardtechniques. Mock cDNA synthesis in the absence of reverse transcriptaseresulted in samples with no detectable PCR amplification of the controlGAPDH or β-actin gene confirming efficient removal of genomic DNAcontamination.

The expression of human PLTR-1 was examined in various human vesselsusing Taqman analysis. The results, set forth below in Table III,indicate that human PLTR-1 is highly expressed in aortic smooth musclecells (SMCs), coronary smooth muscle cells (SMCs), normal artery,interior mammary artery, diseased iliac artery, diseased tibial artery,diseased aorta, and normal saphenous vein. TABLE III Tissue Type Mean β2 Mean ∂∂ Ct Expression  1. Human umbilicial vein 23.27 19.37 3.967.2184    endothelial cells    (HUVECs) - Static  2. HUVECs - Laminar23.23 19.41 3.82 70.8052    shear stress (LSS)  3. Aortic smooth muscle24.77 19.75 5.01 30.9268    cells (SMCs)  4. Coronary SMCs 25.84 20.275.57 21.0505  5. Human adipose tissue 30.41 18.8 11.61 0.3199  6. Normalhuman carotid artery 24.55 18.56 5.99 15.7337  7. Normal human artery26.4 19.64 6.75 9.2585  8. Normal human artery 28.46 19.44 9.02 1.9262 9. Normal human artery 34.9 22.47 12.43 0.1818 10. Internal mammaryartery 29.98 23.05 6.93 8.1725 11. Internal mammary artery 27.82 23.094.72 37.8123 12. Internal mammary artery 29.67 22.57 7.11 7.2641 13.Internal mammary artery 27.91 22.26 5.64 19.9841 14. Internal mammaryartery 26.76 21.31 5.45 22.8763 15. Internal mammary artery 27.21 21.156.07 14.9366 16. Internal mammary artery 33.2 24.45 8.76 2.3146 17.Diseased human iliac artery 26.38 20.27 6.11 14.5282 18. Diseased humantibial artery 23.11 18.15 4.96 32.0174 19. Diseased human aorta 27 20.846.16 14.0333 20. Diseased aorta 28.11 22.31 5.81 17.8244 21. Diseasedaorta 27.75 21.95 5.8 17.9484 22. Diseased aorta 28.28 21.52 6.75 9.258523. Normal human saphenous 28.83 21.2 7.63 5.0658    vein 24. Normalhuman saphenous 23.88 17.48 6.39 11.9239    vein 25. Normal humansaphenous 22.54 16.92 5.62 20.3335    vein 26. Normal human vein 28.0819.19 8.89 2.1079 27. Normal human saphenous 28.11 20.05 8.07 3.7212   vein 28. Normal human vein 26.58 19.2 7.38 6.0243 29. Normal human vein30.28 21.31 8.97 1.9942

Taqman analysis was further used to examine the expression of humanPLTR-1 in human umbilical vein endothelial cells (HUVECs), human aorticendothelial cells (HAECs), and human microvascular endothelial cells(HMVECs) treated with mevastatin for varying amounts of time and atvarying amounts. The results are set forth below in Table IV. Mevastatinis a cholesterol-lowering drug that functions by inhibition of HMG-CoAReductase. As shown below, human PLTR-1 is upregulated by mevastatintreatment, PLTR-1 activity may be useful in screening assays fortherapeutic modulators (e.g., positive modulators). TABLE IVCells/Treatment Mean β 2 Mean ∂∂ Ct Expression HUVEC Vehicle 25.32 19.655.67 19.709 HUVEC Mev 24.11 18.98 5.13 28.6564 HAEC Vehicle 25.06 19.345.72 18.9062 HAEC MEV 26.02 20.98 5.03 30.6069 HMVEC/Vehicle/24 hr 26.3618.12 8.24 3.2962 HMVEC/Mev/24 hr/1X 25.82 18.11 7.71 4.7925HMVEC/MEV/24 HR/2.5X 25.25 18.03 7.22 6.6843 HMVEC/MEV/48 HR/1X 26.1618.61 7.56 5.2992 HMVEC/MEV/48 HR/2.5X 25.19 18.28 6.91 8.3154HUVEC/Vehicle/24 hr 25.2 17.56 7.63 5.0308 HUVEC/Mev/24 hr/1X 24.0718.12 5.95 16.176 HUVEC/MEV/24 HR/2.5X 24.91 18.88 6.04 15.1977HUVEC/MEV/48 HR/1X 26.69 20.66 6.03 15.3566 HUVEC/MEV/48 HR/2.5X 30.0222.24 7.78 4.5655

Example 11 Identification and Characterization of Human TFM-2 and TFM-3cDNAs

In this example, the identification and characterization of the geneencoding human TFM-2 (clone 32146) and human TFM-3 (clone 57259) isdescribed.

Isolation of the Human TFM-2 and TFM-3 cDNAs

The invention is based, at least in part, on the discovery of two humangenes encoding novel polypeptides, referred to herein as human TFM-2 andTFM-3. The entire sequence of the human clone 32146 was determined andfound to contain an open reading frame termed human “TFM-2.” Thenucleotide sequence of the human TFM-2 gene is set forth in the SequenceListing as SEQ ID NO:27. The amino acid sequence of the human TFM-2expression product is set forth in the Sequence Listing as SEQ ID NO:28.The TFM-2 polypeptide comprises 392 amino acids. The coding region (openreading frame) of SEQ ID NO:27 is set forth as SEQ ID NO:29.

The entire sequence of the human clone 57259 was determined and found tocontain an open reading frame termed human “TFM-3.” The nucleotidesequence of the human TFM-3 gene is set forth in the Sequence Listing asSEQ ID NO:30. The amino acid sequence of the human TFM-3 expressionproduct is set forth in the Sequence Listing as SEQ ID NO:31. The TFM-3polypeptide comprises 405 amino acids. The coding region (open readingframe) of SEQ ID NO:30 is set forth as SEQ ID NO:32.

Analysis of the Human TFM-2 and TFM-3 Molecules

A search using the polypeptide sequence of SEQ ID NO:28 was performedagainst the HMM database in PFAM resulting in the identification of apotential monocarboxylate transporter domain in the amino acid sequenceof human TFM-2 at about residues 1-332 of SEQ ID NO:28 (score=35.5), apotential LacY proton/sugar symporter domain in the amino acid sequenceof human TFM-2 at about residues 42-322 of SEQ ID NO:28 (score=−341.8),a potential polysaccharide biosynthesis domain in the amino acidsequence of human TFM-2 at about residues 77-353 of SEQ ID NO:28(score=−96.2), and a potential domain of unknown function, DUF20, in theamino acid sequence of human TFM-2 at about residues 26-326 of SEQ IDNO:28 (score=−133.4).

The amino acid sequence of human TFM-2 was analyzed using the programPSORT (Nakai, K. and Horton, P. (1999) Trends. Biochem. Sci. 24(1)34-35) to predict the localization of the proteins within the cell. Thisprogram assesses the presence of different targeting and localizationamino acid sequences within the query sequence. The results of thisanalysis show that human TFM-2 may be localized to the endoplasmicreticulum, mitochondria or nucleus.

Searches of the amino acid sequence of human TFM-2 were furtherperformed against the Prosite database. These searches resulted in theidentification in the amino acid sequence of human TFM-2 of a potentialglycosaminoglycan attachment site (e.g., at residues 216-219 of SEQ IDNO:28), a number of potential cAMP- and cGMP-dependent protein kinasephosphorylation sites (e.g., at residues 151-154, 385-388 of SEQ IDNO:28), a number of potential protein kinase C phosphorylation sites(e.g., at residues 110-112, 127-129, 134-136, 149-151, 351-353, 361-363of SEQ ID NO:28), a number of potential casein kinase II phosphorylationsites (e.g., at residues 40-43, 134-137, 361-364 of SEQ ID NO:28), anumber of potential N-myristoylation sites (e.g., at residues 17-22,25-30, 32-37, 50-55, 56-61, 77-82, 106-111, 141-146, 176-181, 213-218,260-265, 270-275, 340-345 of SEQ ID NO:28), a potential membranelipoprotein lipid attachment site (e.g., at residues 45-55 of SEQ IDNO:28), and a potential leucine zipper site (e.g., at residues 241-262of SEQ ID NO:28).

A MEMSAT analysis of the polypeptide sequence of SEQ ID NO:28 was alsoperformed (FIG. 17), predicting ten potential transmembrane domains inthe amino acid sequence of human TFM-2 (SEQ ID NO:28) at about residues22-42, 49-69, 76-98, 105-128, 167-186, 207-223, 236-253, 261-285,296-318, and 327-349.

A search of the amino acid sequence of human TFM-2 was also performedagainst the ProDom database. This search resulted in the local alignmentof the human TFM-2 protein with various transporter proteins.

A search using the polypeptide sequence of SEQ ID NO:31 was performedagainst the HMM database in PFAM resulting in the identification of apotential sugar transporter domain in the amino acid sequence of humanTFM-3 at about residues 1-353 of SEQ ID NO:31 (score=−160.9).

The amino acid sequence of human TFM-3 was also analyzed using theprogram PSORT The results of this analysis show that human TFM-3 may belocalized to the endoplasmic reticulum, mitochondria, secretory vesiclesor vacuole.

Searches of the amino acid sequence of human TFM-3 were furtherperformed against the Prosite database. These searches resulted in theidentification in the amino acid sequence of human TFM-3 of a potentialN-glycosylation site (e.g., at residues 348-351 of SEQ ID NO:31), anumber of potential protein kinase C phosphorylation sites (e.g., atresidues 4-6, 85-87, 97-99, 106-108, 129-131, 250-252 of SEQ ID NO:31),a number of potential casein kinase II phosphorylation sites (e.g., atresidues 250-253, 350-353, 373-376, 392-395 of SEQ ID NO:31), a numberof potential N-myristoylation sites (e.g., at residues 15-20, 162-167,246-251, 263-268, 292-297, 382-387, 396-401 of SEQ ID NO:31), a numberof potential amidation sites (e.g., at residues 30-33,209-212 of SEQ IDNO:31), and a number of potential prokaryotic membrane lipoprotein lipidattachment sites (e.g., at residues 189-199, 315-325 of SEQ ID NO:31).

A MEMSAT analysis of the polypeptide sequence of SEQ ID NO:31 was alsoperformed (FIG. 19), predicting nine potential transmembrane domains inthe amino acid sequence of human TFM-3 (SEQ ID NO:31) at about residues7-23, 34-57, 66-82, 150-168, 188-206, 213-237, 255-279, 288-308, and321-337.

A search of the amino acid sequence of human TFM-3 was also performedagainst the ProDom database. This search resulted in the local alignmentof the human TFM-3 protein with various transporter proteins.

Example 12 Identification and Characterization of Human 67118 and 67067cDNAs

In this example, the identification and characterization of the geneencoding human 67118 (clone 67118) and 67067 (clone 67067) is described.

Isolation of the Human 67118 and 67067 cDNAs

The invention is based, at least in part, on the discovery of two humangenes encoding a novel polypeptides, referred to herein as human 67118and 67067. The entire sequence of the human clone 67118 was determinedand found to contain an open reading frame termed human “67118.” Thenucleotide sequence of the human 67118 gene is set forth in the SequenceListing as SEQ ID NO:33. The amino acid sequence of the human 67118expression product is set forth in the Sequence Listing as SEQ ID NO:34.The 67118 polypeptide comprises 1134 amino acids. The coding region(open reading frame) of SEQ ID NO:33 is set forth as SEQ ID NO:35.

The entire sequence of the human clone 67067 was determined and found tocontain an open reading frame termed human “67067.” The nucleotidesequence of the human 67067 gene is set forth in and in the SequenceListing as SEQ ID NO:36. The amino acid sequence of the human 67067expression product is set forth in the Sequence Listing as SEQ ID NO:37.The 67067 polypeptide comprises 1588 amino acids. The coding region(open reading frame) of SEQ ID NO:36 is set forth as SEQ ID NO:38.

Analysis of the Human 67118 and 67067 Molecules

The amino acid sequences of human 67118 and human 67067 were analyzedfor the presence of sequence motifs specific for P-type ATPases (asdefined in Tang, X. et al. (1996) Science 272:1495-1497 and Fagan, M. J.and Saier, M. H. (1994) J. Mol. Evol. 38:57). These analyses resulted inthe identification of a P-type ATPase sequence I motif in the amino acidsequence of human 67118 at residues 179-187 of SEQ ID NO:34 and in theamino acid sequence of human 67067 at residues 175-183 of SEQ ID NO:37.These analyses also resulted in the identification of a P-type ATPasesequence 2 motif in the amino acid sequence of human 67118 at residues411-420 of SEQ ID NO:34. These analyses also resulted in theidentification of a P-type ATPase sequence 2 motif in the amino acidsequence of human 67067 at residues 431-440 of SEQ ID NO:37. Theseanalyses further resulted in the identification of a P-type ATPasesequence 3 motif in the amino acid sequence of human 67118 at residues823-833 of SEQ ID NO:34. These analyses further resulted in theidentification of a P-type ATPase sequence 3 motif in the amino acidsequence of human 67067 at residues 1180-1190 of SEQ ID NO:37.

The amino acid sequences of human 67118 and 67067 were also analyzed forthe presence of phospholipid transporter specific amino acid residues(as defined in Tang, X. et al. (1996) Science 272:1495-1497). Theseanalyses resulted in the identification of phospholipid transporterspecific amino acid residues in the amino acid sequence of human 67118at residues 179, 183, 442, 823, 832, and 833 of SEQ ID NO:34 (FIGS.21A-B). These analyses resulted in the identification of phospholipidtransporter specific amino acid residues 175, 176, 179, 432, 1180, 1189,and 1190 in the amino acid sequence of human 67067 at residues of SEQ IDNO:37 (FIGS. 23A-B).

The amino acid sequences of human 67118 and human 67067 were alsoanalyzed for the presence of extramembrane domains. An N-terminal largeextramembrane domain was identified in the amino acid sequence of human67118 at residues 111-294 of SEQ ID NO:34. A C-terminal largeextramembrane domain was identified in the amino acid sequence of human67118 at residues 369-890 of SEQ ID NO:34. An N-terminal largeextramembrane domain was identified in the amino acid sequence of human67067 at residues 105-286 of SEQ ID NO:37. A C-terminal largeextramembrane domain was identified in the amino acid sequence of human67067 at residues 389-1238 of SEQ ID NO:37.

The amino acid sequence of human 67118 was analyzed using the programPSORT to predict the localization of the proteins within the cell. Thisprogram assesses the presence of different targeting and localizationamino acid sequences within the query sequence. The results of thisanalysis predict that human 67118 may be localized to the endoplasmicreticulum.

Searches of the amino acid sequence of human 67118 were furtherperformed against the Prosite database. These searches resulted in theidentification in the amino acid sequence of human 67118 of a number ofpotential N-glycosylation sites at about residues 397-400, 745-748,921-924, 989-992, and 1001-1004 of SEQ ID NO:34, a number of potentialcAMP-and cGMP-dependent protein kinase phosphorylation sites at aboutresidues 140-143, 558-561, and 705-708 of SEQ ID NO:34, a number ofpotential protein kinase C phosphorylation sites at about residues52-54, 143-145, 169-171, 188-190, 255-257, 259-261, 283-285, 335-337,413-415, 555-557, 714-716, 1017-1019, and 1105-1107 of SEQ ID NO:34, anumber of casein kinase II phosphorylation sites at about residues203-206, 269-272, 287-290, 333-336, 380-383, 418-421, 451-454, 507-510,659-662, 722-725, 910-913, 933-936, and 1103-1106 of SEQ ID NO:34, anumber of potential tyrosine kinase phosphorylation sites at aboutresidues 878-885, 1019-1026 of SEQ ID NO:34, a number ofN-myristoylation sites at about residues 208-213, 498-503, 577-582,762-767, 775-780, 972-977, and 996-1001 of SEQ ID NO:34, an RGD cellattachment sequence at about residues 171-173 of SEQ ID NO:34, and anE1-E2 ATPases phosphorylation site at about residues 414-420 of SEQ IDNO:34.

A MEMSAT analysis of the polypeptide sequence of SEQ ID NO:34 was alsoperformed, predicting ten potential transmembrane domains in the aminoacid sequence of human 67118 (SEQ ID NO:34) at about residues 71-87,94-110, 295-314, 349-368, 891-907, 915-935, 964-987, 1002-1018,1033-1057, and 1064-1088.

A search of the amino acid sequence of human 67118 was also performedagainst the ProDom database, resulting in the identification of severalATPase, hydrolase, and/or transmembrane domain-containing proteins.

The amino acid sequence of human 67067 was analyzed using the programPSORT. The results of this analysis predict that human 67067 may belocalized to the endoplasmic reticulum.

Searches of the amino acid sequence of human 67067 were furtherperformed against the Prosite database. These searches resulted in theidentification in the amino acid sequence of human 67067 of a number ofpotential N-glycosylation sites at about residues 270-273, 340-343,355-358, 1060-1063, 1318-1321, and 1400-1403 of SEQ ID NO:37, aglycosaminoglycan attachment site at about residues 820-823 of SEQ IDNO:37, a number of potential cAMP- and cGMP-dependent protein kinasephosphorylation sites at about residues 447-450, 694-697, 898-901, and1575-1578 of SEQ ID NO:37, a number of protein kinase C phosphorylationsites at about residues 29-31, 45-47, 115-117, 128-130, 247-249,433-435, 473-475, 521-523, 535-537, 555-557, 564-566, 567-569, 579-581,733-735, 737-739, 874-876, 895-897, 949-951, 981-983, 1030-1032,1055-1057, 1475-1477, 1508-1510, 1574-1576, and 1578-1580 of SEQ IDNO:37, a number of potential casein kinase II phosphorylation sites atabout residues 29-32, 128-131, 195-198, 279-282, 342-345, 438-441,457-460, 535-538, 541-544, 607-610, 632-635, 648-651, 666-669, 717-720,743-746, 770-773, 785-788, 797-800, 801-804, 810-813, 824-827, 848-851,972-975, 1014-1017, 1030-1033, 1179-1182, 1200-1203, 1267-1270,1325-1328, 1347-1350, 1500-1503, and 1549-1552 of SEQ ID NO:37, atyrosine kinase phosphorylation site at about residues 1140-1148 of SEQID NO:37, a number of potential N-myristoylation sites at about residues303-308, 453-458, 714-719, 779-784, 798-803, 805-810, 821-826, 880-885,1023-1028, 1196-1201, 1355-1360, and 1501-1506 of SEQ ID NO:37, apotential amidation site at about residues 4-7 of SEQ ID NO:37, anATP/GTP-binding site motif (P-loop) at about residues 1122-1129 of SEQID NO:37, a leucine zipper pattern at about residues 990-1011 of SEQ IDNO:37, and an E1-E2 ATPases phosphorylation site at about residues434-440 of SEQ ID NO:37.

A MEMSAT analysis of the polypeptide sequence of SEQ ID NO:37 was alsoperformed, predicting eight potential transmembrane domains in the aminoacid sequence of human 67067 (SEQ ID NO:37). However, a structural,hydrophobicity, and antigenicity analysis (FIG. 22) resulted in theidentification of ten transmembrane domains. Accordingly, the 67067protein of SEQ ID NO:37 is predicted to have at least ten transmembranedomains, at about residues 65-82, 89-105, 287-304, 366-388, 1239-1259,1322-1343, 1274-1292, 1351-1368, 1377-1399, 1425-1446.

A search of the amino acid sequence of human 67067 was also performedagainst the ProDom database, resulting in the identification of severalATPase, hydrolase, and/or transmembrane domain-containing proteins.

Example 13 Tissue Distribution of 67118 mRNA Using Taqman™ Analysis

This example describes the tissue distribution of human 67118 mRNA in avariety of cells and tissues, as determined using the TaqMan™ procedure.The Taqman™ procedure is a quantitative, reverse transcription PCR-basedapproach for detecting mRNA. The RT-PCR reaction exploits the 5′nuclease activity of AmpliTaq Gold™ DNA Polymerase to cleave a TaqMan™probe during PCR. Briefly, cDNA was generated from the samples ofinterest, including, for example, various normal and diseased vascularand arterial samples, and used as the starting material for PCRamplification. In addition to the 5′ and 3′ gene-specific primers, agene-specific oligonucleotide probe (complementary to the region beingamplified) was included in the reaction (i.e., the Taqman™ probe). TheTaqMan™ probe includes the oligonucleotide with a fluorescent reporterdye covalently linked to the 5′ end of the probe (such as FAM(6-carboxyfluorescein), TET(6-carboxy-4,7,2′,7′-tetrachlorofluorescein), JOE(6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein), or VIC) and aquencher dye (TAMRA (6-carboxy-N,N,N′,N′-tetramethylrhodamine) at the 3′end of the probe.

During the PCR reaction, cleavage of the probe separates the reporterdye and the quencher dye, resulting in increased fluorescence of thereporter. Accumulation of PCR products is detected directly bymonitoring the increase in fluorescence of the reporter dye. When theprobe is intact, the proximity of the reporter dye to the quencher dyeresults in suppression of the reporter fluorescence. During PCR, if thetarget of interest is present, the probe specifically anneals betweenthe forward and reverse primer sites. The 5′-3′ nucleolytic activity ofthe AmpliTaq™ Gold DNA Polymerase cleaves the probe between the reporterand the quencher only if the probe hybridizes to the target. The probefragments are then displaced from the target, and polymerization of thestrand continues. The 3′ end of the probe is blocked to preventextension of the probe during PCR. This process occurs in every cycleand does not interfere with the exponential accumulation of product. RNAwas prepared using the trizol method and treated with DNase to removecontaminating genomic DNA. cDNA was synthesized using standardtechniques. Mock cDNA synthesis in the absence of reverse transcriptaseresulted in samples with no detectable PCR amplification of the controlgene confirms efficient removal of genomic DNA contamination.

The expression levels of human 67118 mRNA in various human cell typesand tissues were analyzed using the Taqman procedure. As shown in TableV, the highest 67118 expression was detected in static Human UmbilicalVein Endothelial Cells (HUVEC), followed by Human Aortic EndothelialCells (HAEC) treated with Mevastatin, HUVEC treated with Mevastatin,HUVEC Vehicle, HUVEC LSS, coronary smooth muscle cells, and aorticsmooth muscle cells. TABLE V Tissue Type Mean β 2 Mean ∂∂ Ct ExpressionAortic SMC 26.02 20.25 5.77 18.3255 Coronary SMC 26.39 20.75 5.6419.9841 Huvec Static 22.2 18.98 3.22 107.3207 Huvec LSS 24.13 18.61 5.5321.7175 H/Adipose/MET 9 32.55 18.07 14.48 0.0438H/Artery/Normal/Carotid/ 33.1 18.61 14.49 0.0435 CLN 595H/Artery/Normal/Carotid/ 35.92 19.82 16.1 0 CLN 598 H/Artery/normal/NDR352 31.34 20.75 10.6 0.6465 H/IM Artery/Normal/AMC 73 39.19 22.92 16.270 H/Muscular Artery/Normal/ 32.06 24.05 8.02 3.8525 AMC 236/ H/MuscularArtery/Normal/ 35.73 22.98 12.75 0 AMC 247/ H/Muscular Artery/Normal/32.99 22.48 10.52 0.6834 AMC 254/ H/Muscular Artery/Normal/ 30.56 21.329.23 1.6595 AMC 259/ H/Muscular Artery/Normal/ 31.06 21.65 9.4 1.4751AMC 261/ H/Muscular Artery/Normal/ 30.89 23.39 7.5 5.5243 AMC 275/H/Aorta/Diseased/PIT 732 32.84 21.31 11.54 0.337 H/Aorta/Diseased/PIT710 30.74 22.4 8.35 3.0754 H/Aorta/Diseased/PIT 711 30.75 22.13 8.622.5417 H/Aorta/Diseased/PIT 712 29.51 21.91 7.61 5.1365H/Artery/Diseased/iliac/ 27.44 18.02 9.41 1.4649 NDR 753H/Artery/Diseased/Tibial/ 33.13 19.41 13.72 0.0744 PIT 679H/Vein/Normal/SaphenousAMC 30.36 20.02 10.34 0.7715 107H/Vein/Normal/NDR 239 37.15 20.83 16.32 0 H/Vein/Normal/Saphenous/ 31.220 11.21 0.4236 NDR 237 H/Vein/Normal/PIT 1010 27.36 20.09 7.27 6.4791H/Vein/Normal/AMC 191 29.32 21.59 7.73 4.7102 H/Vein/Normal/AMC 13028.72 20.66 8.06 3.7342 H/Vein/Normal/AMC 188 31.63 24.34 7.28 6.4343HUVEC Vehicle 25.46 19.84 5.63 20.2631 HUVEC Mev 24.61 19.27 5.3424.6034 HAEC Vehicle 25.65 20 5.65 19.915 HAEC Mev 26.72 21.76 4.9632.1286

Example 14 Tissue Distribution of 67067 mRNA Using Taqman™ Analysis

The tissue distribution of human 67067 mRNA in a variety of cells andtissues was determined using the TaqMan™ procedure, as described above.

As shown in Table VI, below, 67067 is overexpressed in colon tumortissue as compared to normal tumor tissue, indicating a possible rolefor 67067 in cellular proliferation disorders, e.g., cancer, including,but not limited to colon cancer. Human 67067 mRNA is also highlyexpressed in normal brain cortex tissue and normal ovary, for example.TABLE VI Tissue Type Mean β 2 Mean ∂∂ Ct Expression Artery normal 32.9522.56 10.4 0.7401 Aorta diseased 34.75 23.2 11.55 0.3335 Vein normal38.53 21.36 17.18 0 Coronary SMC 38.67 22.54 16.14 0 HUVEC 39.28 22.7916.49 0 Hemangioma 33.84 21.3 12.54 0.1679 Heart normal 36.09 21.0515.04 0 Heart CHF 35.33 21.5 13.82 0 Kidney 31.6 21.34 10.26 0.8155Skeletal Muscle 36.3 23.51 12.79 0 Adipose normal 40 23.07 16.93 0Pancreas 31.49 23.73 7.76 4.5973 primary osteoblasts 40 21.06 18.95 0Osteoclasts (diff) 35.04 18.19 16.85 0 Skin normal 34.23 23.73 10.510.6858 Spinal cord normal 30.47 22.32 8.14 3.5327 Brain Cortex normal28.66 23.72 4.95 32.4643 Brain Hypothalamus 30.32 24.07 6.25 13.139normal Nerve 30.95 22.55 8.4 2.9501 DRG (Dorsal Root 30.07 22.88 7.26.8248 Ganglion) Breast normal 37.3 22.5 14.8 0 Breast tumor 36.56 22.3814.19 0 Ovary normal 27.73 21.25 6.47 11.2807 Ovary Tumor 31.93 20.5711.36 0.3805 Prostate Normal 37.28 19.95 17.34 0 Prostate Tumor 33.8721.14 12.73 0.1472 Salivary glands 32.1 20.75 11.35 0.3831 Colon normal27.24 20.11 7.13 7.1146 Colon Tumor 26.34 22.9 3.44 91.823 Lung normal35.78 19.95 15.84 0 Lung tumor 28.48 20.66 7.82 4.4253 Lung COPD 36.0119.41 16.61 0 Colon IBD 25.16 19.02 6.14 14.18 Liver normal 37.01 21.5815.43 0 Liver fibrosis 35.28 22.5 12.79 0 Spleen normal 38.06 19.9818.08 0 Tonsil normal 28.32 18.69 9.63 1.2621 Lymph node normal 34.8820.49 14.39 0.0467 Small intestine normal 28.99 21.86 7.13 7.1641Macrophages 36.06 18.16 17.89 0 Synovium 34.62 21.27 13.35 0.0958 BM-MNC40 20.75 19.25 0 Activated PBMC 36.87 18.41 18.47 0 Neutrophils 40 19.5920.41 0 Megakaryocytes 37.98 20 17.98 0 Erythroid 40 23.07 16.93 0positive control 29.45 21.89 7.57 5.2809

Example 15 Identification and Characterization of Human 62092 cDNA

In this example, the identification and characterization of the geneencoding human 62092 (clone 62092) is described.

Isolation of the Human 62092 cDNA

The invention is based, at least in part, on the discovery of genesencoding novel members of the histidine triad family. The entiresequence of human clone Fbh62092 was determined and found to contain anopen reading frame termed human “62092”.

The nucleotide sequence encoding the human 62092 is set forth as SEQ IDNO:39. The protein encoded by this nucleic acid comprises about 163amino acids and has the amino acid sequence set forth as SEQ ID NO:40.The coding region (open reading frame) of SEQ ID NO:39 is set forth asSEQ ID NO:41.

Analysis of the Human 62092 Molecules

The amino acid sequence of human 62092 was analyzed using the programPSORT to predict the localization of the proteins within the cell. Thisprogram assesses the presence of different targeting and localizationamino acid sequences within the query sequence. The results of theanalyses show that human 62092 is most likely localized to themitochondria.

Searches of the amino acid sequence of human 62092 were also performedagainst the HMM database. These searches resulted in the identificationof a “HIT family domain” at about residues 54-155 (score=180.3).

Searches of the amino acid sequence of human 62092 were furtherperformed against the Prosite™ database. These searches resulted in theidentification of a “HIT family signature motif” at about residues136-151 of SEQ ID NO:40. These searches further resulted in theidentification in the amino acid sequence of human 62092 of a potentialprotein kinase C phosphorylation site at about residues 121-123 of SEQID NO:40, a potential casein kinase II phosphorylation site at aboutresidues 101-104 of SEQ ID NO:40, and a number of N-myristoylation sitesat about residues 10-15, 22-27, 33-38, 50-55, and 126-131 of SEQ IDNO:40.

A search of the amino acid sequence of human 62092 was also performedagainst the ProDom database, resulting in the identification of a“protein HIT-like domain” at amino acid residues 54-155 of SEQ ID NO:40.

Example 16 Tissue Distribution of 62092 mRNA Using Taqman™ Analysis

The tissue distribution of human 62092 mRNA in a variety of cells andtissues was determined using the TaqMan™ procedure, as described above.

As shown in Table VII, below, 62092 is notably overexpressed in lungtumor tissue as compared to normal lung tissue, indicating a possiblerole for 62092 in cellular proliferation disorders, e.g., cancer,including, but not limited to lung cancer. Human 62092 mRNA is alsohighly expressed in activated PMBC, erythroid cells, normal brain cortexand hypothalamus, and normal liver tissue, for example. TABLE VII TissueType Mean β 2 Mean ∂∂ Ct Expression Artery normal 28.59 22.41 4.254.5983 Aorta diseased 30.42 23.1 5.34 24.7745 Vein normal 28.43 20.75.74 18.7106 Coronary SMC 28.11 23.03 3.1 117.034 HUVEC 27.15 22.81 2.36195.4674 Hemangioma 28.3 20.66 5.66 19.8461 Heart normal 27.23 20.694.55 42.6888 Heart CHF 26.16 21.15 3.02 123.2791 Kidney 26.25 21.32 2.94130.3082 Skeletal Muscle 27.87 23.18 2.7 153.8931 Adipose normal 28.2422.71 3.54 85.6739 Pancreas 28.21 23.65 2.58 167.2409 primaryosteoblasts 30.15 21.09 7.08 7.3911 Osteoclasts (diff) 27.38 18.06 7.346.1936 Skin normal 30 23.63 4.39 47.6956 Spinal cord normal 29.34 22.315.04 30.2903 Brain Cortex normal 28.2 25.26 0.95 515.8416 BrainHypothalamus normal 27.98 23.97 2.02 246.5582 Nerve 29.12 22.73 4.4147.039 DRG (Dorsal Root Ganglion) 27.6 22.63 2.98 126.3064 Breast normal28.19 22.42 3.79 72.544 Breast tumor 30.18 22.86 5.33 24.8605 Ovarynormal 27.4 21.17 4.24 52.9216 Ovary Tumor 26.63 20.82 3.83 70.3162Prostate Normal 26.62 19.69 4.95 32.4643 Prostate Tumor 26.46 21.15 3.3399.4421 Salivary glands 27.92 20.61 5.33 24.8605 Colon normal 26.4320.09 4.36 48.8669 Colon Tumor 28.53 22.93 3.61 81.8996 Lung normal 27.119.63 5.49 22.328 Lung tumor 24.89 23.47 −0.56 1479.3875 Lung COPD 26.1819.24 4.96 32.1286 Colon IBD 26.08 18.84 5.25 26.1871 Liver normal 25.4821.27 2.22 214.6414 Liver fibrosis 27.26 22.46 2.81 142.1021 Spleennormal 28.93 19.84 7.11 7.2641 Tonsil normal 26.32 18.84 5.5 22.0971Lymph node normal 28.49 20.27 6.24 13.2304 Small intestine normal 28.9121.65 5.28 25.8266 Macrophages 32.22 18.07 12.16 0.2185 Synovium 30.8621.7 7.18 6.8961 BM-MNC 32.14 20.59 9.56 1.3248 Neutrophils 27.84 19.346.52 10.8964 Megakaryocytes 24.32 19.77 2.57 168.4042 Erythroid 26.6823.36 1.33 397.7682 Activated PBMC 28.11 26.91 −0.79 1723.0923 positivecontrol 26.71 21.86 2.87 137.2616

Example 17 Tissue Distribution of 67118,67067, and 62092 mRNA Using insitu Analysis

This example describes the tissue distribution of human 67118, 67067,and/or 62092 mRNA, as may be determined using in situ hybridizationanalysis. For in situ analysis, various tissues are first frozen on dryice. Ten-micrometer-thick sections of the tissues are postfixed with 4%formaldehyde in DEPC-treated 1× phosphate-buffered saline at roomtemperature for 10 minutes before being rinsed twice in DEPC 1×phosphate-buffered saline and once in 0.1 M triethanolamine-HCl (pH8.0). Following incubation in 0.25% acetic anhydride-0.1 Mtriethanolamine-HCl for 10 minutes, sections are rinsed in DEPC 2×SSC(1×SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Tissue is thendehydrated through a series of ethanol washes, incubated in 100%chloroform for 5 minutes, and then rinsed in 100% ethanol for 1 minuteand 95% ethanol for 1 minute and allowed to air dry.

Hybridizations are performed with ³⁵S-radiolabeled (5×10⁷ cpm/ml) cRNAprobes. Probes are incubated in the presence of a solution containing600 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 0.01% sheared salmon spermDNA, 0.01% yeast tRNA, 0.05% yeast total RNA type X1, 1× Denhardt'ssolution, 50% formamide, 10% dextran sulfate, 100 mM dithiothreitol,0.1% sodium dodecyl sulfate (SDS), and 0.1% sodium thiosulfate for 18hours at 55° C.

After hybridization, slides are washed with 2×SSC. Sections are thensequentially incubated at 37° C. in TNE (a solution containing 10 mMTris-HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA), for 10 minutes, in TNEwith 10 μg of RNase A per ml for 30 minutes, and finally in TNE for 10minutes. Slides are then rinsed with 2×SSC at room temperature, washedwith 2×SSC at 50° C. for 1 hour, washed with 0.2×SSC at 55° C. for 1hour, and 0.2×SSC at 60° C. for 1 hour. Sections are then dehydratedrapidly through serial ethanol-0.3 M sodium acetate concentrationsbefore being air dried and exposed to Kodak Biomax MR scientific imagingfilm for 24 hours and subsequently dipped in NB-2 photoemulsion andexposed at 4° C. for 7 days before being developed and counter stained.

Example 18 Detection of 67118,67067, and 62092 Transcripts and Structureby RT-PCR Analysis

This example describes a method for determining the structure andexpression level of human 67118, 67067, or 62092, as may be determinedusing RT-PCR analysis. For RT-PCR analysis, total RNA is first isolatedfrom various tissues. Total RNA is reverse-transcribed usingoligodeoxythymidylate primers and the resulting single-stranded cDNAproducts used as templates for first round PCR amplification. Firstround PCR amplification is performed using primers designed using the67118, 67067, or 62092 sequence set forth as SEQ ID NO:33, 36, ot 39,respectively. Second round PCR amplification is performed using nestedprimers derived from the 67118, 67067, or 62092 sequence (SEQ ID NO:33,36, or 39, respectively). Amplification products are electrophoresed inagarose gels and detected by ethidium bromide staining.

Quantitation of the signal generated by RT-PCR analysis gives a measureof the expression level of human 67118, 67067, or 62092.

The structure of human 67118, 67067, or 62092 can be determined byexcising the RT-PCR product from an agarose gel, purifying it, andsequencing it to determine if there are missense or point mutations, orif there is a deletion within the human 67118, 67067, or 62092 gene.

Example 19 Identification and Characterization of Human HAAT cDNA

In this example, the identification and characterization of the geneencoding human HAAT (clone Fbh58295FL) is described.

Isolation of the Human HAAT cDNA

The invention is based, at least in part, on the discovery of genesencoding novel members of the amino acid transporter family. The entiresequence of human clone Fbh58295FL was determined and found to containan open reading frame termed human “HAAT”.

The nucleotide sequence encoding the human HAAT is set forth as SEQ IDNO:51. The protein encoded by this nucleic acid comprises about 485amino acids and has the amino acid sequence set forth as SEQ ID NO:52.The coding region (open reading frame) of SEQ ID NO:51 is set forth asSEQ ID NO:53.

Analysis of the Human HAAT Molecules

The HAAT amino acid sequence (SEQ ID NO:52) was aligned with the aminoacid sequence of the rat amino acid system A transporter (ratATA2) usingthe CLUSTAL W (1.74) multiple sequence alignment program.

An analysis of the amino acid sequence of HAAT was performed usingMEMSAT. This analysis resulted in the identification of 10 possibletransmembrane domains in the amino acid sequence of HAAT at residues68-72, 135-156, 190-207, 214-232, 256-274, 287-308, 334-356, 373-390,397-421, and 435-453 of SEQ ID NO:52 (FIG. 28). An additional predictedtransmembrane domain (i.e., TM1 is also shown.)

A search using the polypeptide sequence of SEQ ID NO:52 was performedagainst the HMM database in PFAM resulting in the identification of atransmembrane amino acid transporter domain in the amino acid sequenceof HAAT at about residues 64 to 445 of SEQ ID NO:52 (score=187.2).

The amino acid sequence of HAAT was further analyzed using the programPSORT (which can be found on the National Institute for Basic Biologyweb site) to predict the localization of the proteins within the cell.This program assesses the presence of different targeting andlocalization amino acid sequences within the query sequence. The resultsof the analysis show that HAAT is most likely localized to theendoplasmic reticulum.

To further identify potential structural and/or functional properties ina protein of interest, the amino acid sequence of the protein issearched against a database of annotated protein domains (e.g., theProDom database) using the default parameters (available athttp://www.toulouse.inra.fr/prodom.html). A search of the amino acidsequence of HAAT (SEQ ID NO:52) was performed against the ProDomdatabase. This search resulted in the local alignment of the HAATprotein with various C. Elegans and/or amino acid proteintransporter/permease proteins. Specifically, amino acid residues288-456, 136-300, and 35-325 of SEQ ID NO:52 have significant identityto various C. elegans-related proteins. Amino acid residues 36-346 ofSEQ ID NO:52 have significant identity to various amino acid proteintransporter/permease-related proteins.

A search of the amino acid sequence of HAAT (SEQ ID NO:52) was performedagainst the Prosite database. These searches resulted in theidentification in the amino acid sequence of HAAT of a number ofpotential glycosylation sites, e.g., at amino acid residues 175-178,221-224, 434-437, and 476-479; a potential cAMP and cGMP-dependentprotein kinase phosphorylation site, e.g., at amino acid residues103-106; a number of potential protein kinase C phosphorylation sites,e.g., at amino acid residues 281-283, 331-333, 360-362, and 460-462; anumber of potential casein kinase II phosphorylation sites, e.g., atamino acid residues 16-19, 134-137, and 452-455; a potential tyrosinekinase phosphorylation site, e.g., at amino acid residues 185-193; and anumber of potential N-myristoylation sites, e.g., at amino acid residues52-57, 60-65, 293-298, 339-344, 401-406, and 448-453.

Tissue Distribution of HAAT mRNA

This example describes the tissue distribution of human HAAT mRNA, asmay be determined using in situ hybridization analysis. For in situanalysis, various tissues, e.g. tissues obtained from brain, are firstfrozen on dry ice. Ten-micrometer-thick sections of the tissues arepostfixed with 4% formaldehyde in DEPC-treated 1× phosphate-bufferedsaline at room temperature for 10 minutes before being rinsed twice inDEPC 1× phosphate-buffered saline and once in 0.1 M triethanolamine-HCl(pH 8.0). Following incubation in 0.25% acetic anhydride-0.1 Mtriethanolamine-HCl for 10 minutes, sections are rinsed in DEPC 2×SSC(1×SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Tissue is thendehydrated through a series of ethanol washes, incubated in 100%chloroform for 5 minutes, and then rinsed in 100% ethanol for 1 minuteand 95% ethanol for 1 minute and allowed to air dry.

Hybridizations are performed with ³⁵S-radiolabeled (5×10⁷ cpm/ml) cRNAprobes. Probes are incubated in the presence of a solution containing600 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 0.01% sheared salmon spermDNA, 0.01% yeast tRNA, 0.05% yeast total RNA type X1, 1× Denhardt'ssolution, 50% formamide, 10% dextran sulfate, 100 mM dithiothreitol,0.1% sodium dodecyl sulfate (SDS), and 0.1% sodium thiosulfate for 18hours at 55° C.

After hybridization, slides are washed with 2×SSC. Sections are thensequentially incubated at 37° C. in TNE (a solution containing 10 mMTris-HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA), for 10 minutes, in TNEwith 10 μg of RNase A per ml for 30 minutes, and finally in TNE for 10minutes. Slides are then rinsed with 2×SSC at room temperature, washedwith 2×SSC at 50° C. for 1 hour, washed with 0.2×SSC at 55° C. for 1hour, and 0.2×SSC at 60° C. for 1 hour. Sections are then dehydratedrapidly through serial ethanol-0.3 M sodium acetate concentrationsbefore being air dried and exposed to Kodak Biomax MR scientific imagingfilm for 24 hours and subsequently dipped in NB-2 photoemulsion andexposed at 4° C. for 7 days before being developed and counter stained.

Example 20 Tissue Expression Analysis of HAAT mRNA Using Taqman Analysis

This example describes the tissue distribution of HAAT in a variety ofcells and tissues, as determined using the TaqMan™ procedure. TheTaqman™ procedure is a quantitative, reverse transcription PCR-basedapproach for detecting mRNA. The RT-PCR reaction exploits the 5′nuclease activity of AmpliTaq Gold™ DNA Polymerase to cleave a TaqMan™probe during PCR. Briefy, cDNA was generated from the samples ofinterest, including, for example, various normal and diseased vascularand arterial samples, and used as the starting material for PCRamplification. In addition to the 5′ and 3′ gene-specific primers, agene-specific oligonucleotide probe (complementary to the region beingamplified) was included in the reaction (i.e., the Taqman™ probe). TheTaqMan™ probe includes the oligonucleotide with a fluorescent reporterdye covalently linked to the 5′ end of the probe (such as FAM(6-carboxyfluorescein), TET(6-carboxy-4,7,2′,7′-tetrachlorofluorescein), JOE(6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein), or VIC) and aquencher dye (TAMRA (6-carboxy-N,N,N′,N′-tetramethylrhodamine) at the 3′end of the probe.

During the PCR reaction, cleavage of the probe separates the reporterdye and the quencher dye, resulting in increased fluorescence of thereporter. Accumulation of PCR products is detected directly bymonitoring the increase in fluorescence of the reporter dye. When theprobe is intact, the proximity of the reporter dye to the quencher dyeresults in suppression of the reporter fluorescence. During PCR, if thetarget of interest is present, the probe specifically anneals betweenthe forward and reverse primer sites. The 5′-3′ nucleolytic activity ofthe AmpliTaq™ Gold DNA Polymerase cleaves the probe between the reporterand the quencher only if the probe hybridizes to the target. The probefragments are then displaced from the target, and polymerization of thestrand continues. The 3′ end of the probe is blocked to preventextension of the probe during PCR. This process occurs in every cycleand does not interfere with the exponential accumulation of product. RNAwas prepared using the trizol method and treated with DNase to removecontaminating genomic DNA. cDNA was synthesized using standardtechniques. Mock cDNA synthesis in the absence of reverse transcriptaseresulted in samples with no detectable PCR amplification of the controlgene confirms efficient removal of genomic DNA contamination.

The expression levels of HAAT mRNA in various human cell types andtissues were analyzed using the Taqman procedure. As shown in TableVIII, the highest HAAT expression was detected in brain cortex and brainhypothalamus, followed by Human Umbilical Vein Endothelial Cells(HUVEC), followed by lung tumor cells. TABLE VIII Tissue Type Mean β 2Mean ∂∂ Ct Expression Artery normal 32.62 21.77 10.84 0.5456 Aortadiseased 35.84 22.43 13.41 0 Vein normal 34.13 20.47 13.65 0.0775Coronary SMC 30.76 21.59 9.17 1.736 HUVEC 29.41 21.81 7.6 5.1543Hemangioma 35.07 20.97 14.1 0 Heart normal 32.7 20.89 11.81 0.2795 HeartCHF 33.63 21.02 12.62 0.1594 Kidney 31.55 20.51 11.04 0.4749 SkeletalMuscle 35.09 22.86 12.22 0 Adipose normal 37.84 22.04 15.81 0 Pancreas33.67 23.13 10.55 0.6693 primary osteoblasts 32 20.4 11.6 0.3233Osteoclasts (diff) 33.98 17.84 16.15 0.0138 Skin normal 36.29 22.2 14.10 Spinal cord normal 32.73 21.68 11.05 0.4716 Brain Cortex normal 28.9523.01 5.95 16.2322 Brain Hypothalamus normal 30 23.47 6.53 10.8212 Nerve33.59 21.82 11.77 0.2873 DRG (Dorsal Root Ganglion) 31.25 21.5 9.761.1573 Breast normal 34.73 21.56 13.18 0.1081 Breast tumor 34.16 21.512.66 0.154 Ovary normal 32.03 20.81 11.23 0.4178 Ovary Tumor 36.33 19.516.82 0 Prostate Normal 32.02 19.65 12.37 0.1896 Prostate Tumor 33.3620.43 12.93 0.1281 Salivary glands 36.17 20.1 16.07 0 Colon normal 36.3319.33 17 0 Colon Tumor 36.05 22.23 13.82 0 Lung normal 34.79 19.38 15.410.023 Lung tumor 28.02 20.03 7.99 3.9471 Lung COPD 33.26 18.61 14.650.039 Colon IBD 34.37 18.07 16.3 0.0124 Liver normal 33.95 20.64 13.320.0981 Liver fibrosis 35.04 21.56 13.48 0 Spleen normal 35.76 19.4316.34 0 Tonsil normal 32.28 18.5 13.79 0.0708 Lymph node normal 34.3120.06 14.25 0.0513 Small intestine normal 35.59 20.93 14.65 0Macrophages 31.75 17.61 14.14 0.0556 Synovium 37.21 21.02 16.2 0 BM-MNC32.71 20.16 12.55 0.1673 Activated PBMC 31.84 18.16 13.69 0.0759Neutrophils 28.14 18.25 9.89 1.0539 Megakaryocytes 32.52 19.1 13.430.0909 Erythroid 32.9 21.09 11.81 0.2795 positive control 30.11 20.979.15 1.7603

Example 21 Identification and Characterization of Human HST-4 and HST-5cDNAs

In this example, the identification and characterization of the geneencoding human HST-4 (clone 57255FL) and HST-5 (clone 57255alt) isdescribed.

Isolation of the Human HST-4 and HST-5 cDNAs

The invention is based, at least in part, on the discovery of a humangene encoding a novel polypeptide, referred to herein as human HST-4.The entire sequence of the human clone 57255FL was determined and foundto contain an open reading frame termed human “HST-4.” The nucleotidesequence of the human HST-4 gene is set forth in the Sequence Listing asSEQ ID NO:54. The amino acid sequence of the human HST-4 expressionproduct is set forth in the Sequence Listing as SEQ ID NO:55. The HST-4polypeptide comprises 438 amino acids. The coding region (open readingframe) of SEQ ID NO:54 is set forth as SEQ ID NO:56. The HST-4 proteinis predicted to contain a signal peptide of 43 residues in theamino-terminal end, which would be cleaved off to result in a maturepeptide comprising amino acid residues 44-438 of SEQ I) NO:55.

The invention is further based, at least in part, on the discovery of ahuman gene encoding a novel polypeptide, referred to herein as humanHST-5. The entire sequence of the human clone 57255alt was determinedand found to contain an open reading frame termed human “HST-5.” Thenucleotide sequence of the human HST-5 gene is set forth in the SequenceListing as SEQ ID NO:57. The amino acid sequence of the human HST-5expression product is set forth in the Sequence Listing as SEQ ID NO:58.The HST-5 polypeptide comprises 436 amino acids. The coding region (openreading frame) of SEQ ID NO:57 is set forth as SEQ ID NO:59. The HST-5protein is predicted to contain a signal peptide of 43 residues in theamino-terminal end, which would be cleaved off to result in a maturepeptide comprising amino acid residues 44-436 of SEQ ID NO:58.

HST-4 and HST-5 are splice variants. Splice variants are variants whichresult from alternative splicing of the same gene.

Analysis of the Human HST-4 and HST-5 Molecules HST-4

The amino acid sequence of human HST-4 (SEQ ID NO:55) was analyzed usingthe program PSORT (www.psort.nibb.ac.jp) to predict the localization ofthe proteins within the cell. This program assesses the presence ofdifferent targeting and localization amino acid sequences within thequery sequence. The results of this analysis show that human HST-4 maybe localized to the endoplasmic reticulum and mitochondria.

A search using the polypeptide sequence of SEQ ID NO:55 was performedagainst the HMM database in PFAM resulting in the identification of asugar transporter family domain in the amino acid sequence of humanHST-4 at about residues 25-418 of SEQ ID NO:55 (score=−210.9), and amonocarboxylate transporter family domain in the amino acid sequence ofhuman HST-4 at about residues 23-431 of SEQ ID NO:55 (score=−144.9).

Searches of the amino acid sequence of human HST-4 were furtherperformed against the Prosite database. These searches resulted in theidentification in the amino acid sequence of human HST-4 of a potentialN-glycosylation site, a number of potential protein kinase Cphosphorylation sites, a number of potential casein kinase IIphosphorylation sites, a number of potential N-myristoylation sites, anda potential sugar transport protein signature 2.

A MEMSAT analysis of the polypeptide sequence of SEQ ID NO:55 was alsoperformed, predicting ten transmembrane domains in the amino acidsequence of human HST-4 (SEQ ID NO:55) at about amino acid residues25-49, 62-80, 92-113, 126-143, 154-178, 186-202, 278-298, 318-337,372-395, and 402-423. This protein was also predicted to contain asignal peptide of 43 residues in the amino-terminal end, which would becleaved off to result in a mature peptide comprising amino acid residues44-438 of SEQ ID NO:55. A MEMSAT analysis of the presumed maturepolypeptide sequence was also performed, predicting nine transmembranedomains in the mature amino acid sequence of HST-4 at about amino acidresidues 63-81, 93-114, 127-144, 155-179, 187-203, 279-299, 319-338,373-396, and 403-424 of SEQ ID NO:55.

A search of SEQ ID NO:55 was also performed against the ProDom database.The results of this search identified matches against protein domainsdescribed as “Polyphosphate IPP Inositol 1-Phosphatase”, “RelatedPermease Transport Membrane”, “NPT 1(3) Transport PhosphateCotransporter Renal Na-Dependent Inorganic Glycoprotein Transmembrane”,“GUDP (2) Transmembrane Transport Transporter Permease” and the like.

HST-5

The amino acid sequence of human HST-5 (SEQ ID NO:58) was analyzed usingthe program PSORT (www.psort.nibb.ac.jp) to predict the localization ofthe proteins within the cell. The results of this analysis show thathuman HST-5 may be localized to the endoplasmic reticulum, vacuoles,mitochondria, Golgi, and cytoplasm.

Searches of the amino acid sequence of human HST-5 (SEQ ID NO:58) wereperformed against the Prosite database. These searches resulted in theidentification in the amino acid sequence of human HST-5 of a potentialN-glycosylation site, a potential cAMP- and cGMP-dependent proteinkinase C phosphorylation site, a number of potential protein kinase Cphosphorylation sites, a number of potential casein kinase IIphosphorylation sites, a number of potential N-myristoylation sites, aprokaryotic membrane lipoprotein lipid attachment site, and a sugartransport protein signature 2.

A search using the polypeptide sequence of SEQ ID NO:58 was performedagainst the HMM database in PFAM resulting in the identification of asugar transporter family domain in the amino acid sequence of humanHST-5 at about residues 23-429 of SEQ ID NO:58 (score=−139.4), and amonocarboxylate transporter family domain in the amino acid sequence ofhuman HST-5 at about residues 25-416 of SEQ ID NO:58 (score=−200.0).

A MEMSAT analysis of the polypeptide sequence of SEQ ID NO:58 was alsoperformed, predicting eleven transmembrane domains in the amino acidsequence of human HST-5 (SEQ ID NO:58) at about amino acid residues30-51, 62-84, 92-111, 126-143, 154-178, 186-202, 240-260, 276-296,316-335, 370-393, and 400-421. This protein was also predicted tocontain a signal peptide of 43 residues in the amino-terminal end, whichwould be cleaved off to result in a mature peptide comprising amino acidresidues 44-436 of SEQ ID NO:58. A MEMSAT analysis of the presumedmature polypeptide sequence was also performed, predicting tentransmembrane domains in the mature amino acid sequence of HST-5 atabout residues 63-85, 93-112, 127-144, 155-179, 187-203, 241-261,277-297, 317-336, 371-394 and 401-422 of SEQ ID NO:58.

A search of SEQ ID NO:58 was also performed against the ProDom database.The results of this search identified matches against protein domainsdescribed as “Polyphosphate IPP Inositol 1-Phosphatase”, “RelatedPermease Transport Membrane”, “NPT 1(3) Transport PhosphateCotransporter Renal Na-Dependent Inorganic Glycoprotein Transmembrane”,“GUDP (2) Transmembrane Transport Transporter Permease” and the like.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated nucleic acid molecule selected from the group consistingof: (a) a nucleic acid molecule comprising a nucleotide sequence whichis at least 90% identical to the nucleotide sequence of SEQ ID NO:1, 3,4, 6, 7, 9, 12, 14, 15, 17, 19, 21, 27, 29, 30, 32, 33, 35, 36, 38, 39,41, 51, 53, 54, 56, 57, or 59, or a complement thereof; (b) a nucleicacid molecule comprising a fragment of at least 30 nucleotides of anucleic acid comprising the nucleotide sequence of SEQ ID NO:1, 3, 4, 6,7, 9, 12, 14, 15, 17, 19, 21, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41,51, 53, 54, 56, 57, or 59, or a complement thereof; (c) a nucleic acidmolecule which encodes a polypeptide comprising an amino acid sequenceat least about 90% identical to the amino acid sequence of SEQ ID NO:2,5, 8, 13, 16, 20, 28, 31, 34, 37, 40, 52, 55 or 58, or a complementthereof; (d) a nucleic acid molecule which encodes a naturally-occurringallelic variant polypeptide comprising the amino acid sequence set forthin SEQ ID NO: 2, 5, 8, 13, 16, 20, 28, 31, 34, 37, 40, 52, 55 or 58, ora complement thereof; and (e) a nucleic acid molecule which encodes afragment of a polypeptide comprising the amino acid sequence of SEQ IDNO:2, 5, 8, 13, 16, 20, 28, 31, 34, 37, 40, 52, 55 or 58, wherein thefragment comprises at least 10 contiguous amino acid residues of theamino acid sequence of SEQ ID NO:2, 5, 8, 13, 16, 20, 28, 31, 34, 37,40, 52, 55 or 58, or a complement thereof.
 2. The isolated nucleic acidmolecule of claim 1, further comprising a nucleotide sequence encoding aheterologous polypeptide.
 3. A vector comprising the nucleic acidmolecule of claim
 1. 4. The vector of claim 3, which is an expressionvector.
 5. A host cell transfected with the expression vector of claim4.
 6. A method of producing a polypeptide comprising culturing the hostcell of claim 5 in an appropriate culture medium to, thereby, producethe polypeptide.
 7. The isolated nucleic acid molecule of claim 1,selected from the group consisting of: (a) a nucleic acid moleculecomprising the nucleotide sequence set forth in SEQ ID NO:1, 4, 7, 12,15, 19, 27, 30, 33, 36, 39, 51, 54 or 57, or a complement thereof; (b) anucleic acid molecule comprising the nucleotide sequence set forth inSEQ ID NO:3, 6, 9, 14, 17, 21, 29, 32, 35, 38, 41, 53, 56, or 59, or acomplement thereof; and (c) a nucleic acid molecule which encodes apolypeptide comprising the amino acid sequence set forth in SEQ ID NO:2,5, 8, 13, 16, 20, 28, 31, 34, 37, 40, 52, 55 or 58, or a complementthereof.
 8. An isolated polypeptide selected from the group consistingof: (a) a polypeptide which is encoded by a nucleic acid moleculecomprising a nucleotide sequence which is at least 90% identical to anucleic acid comprising the nucleotide sequence of SEQ ID NO:1, 3, 4, 6,7, 9, 12, 14, 15, 17, 19, 21, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41,51, 53, 54, 56, 57, or 59; (b) a fragment of a polypeptide comprisingthe amino acid sequence of SEQ ID NO:2, 5, 8, 13, 16, 20, 28, 31, 34,37, 40, 52, 55 or 58, wherein the fragment comprises at least 10contiguous amino acids of SEQ ID NO:2, 5, 8, 13, 16, 20, 28, 31, 34, 37,40, 52, 55 or 58; (c) a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:2, 5, 8, 13,16, 20, 28, 31, 34, 37, 40, 52, 55 or 58, wherein the polypeptide isencoded by a nucleic acid molecule which hybridizes to complement of anucleic acid molecule consisting of SEQ ID NO:1, 3, 4, 6, 7, 9, 12, 14,15, 17, 19, 21, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 51, 53, 54, 56,57, or 59 under stringent conditions; and d) a polypeptide comprising anamino acid sequence which is at least 90% identical to the amino acidsequence of SEQ ID NO:2, 5, 8, 13, 16, 20, 28, 31, 34, 37, 40, 52, 55 or58.
 9. The polypeptide of claim 8, further comprising heterologous aminoacid sequences.
 10. An antibody which selectively binds to a polypeptideof claim
 8. 11. A method for detecting the presence of a polypeptide ofclaim 8 in a sample comprising: a) contacting the sample with a compoundwhich selectively binds to the polypeptide; and b) determining whetherthe compound binds to the polypeptide in the sample to thereby detectthe presence of a polypeptide of claim 8 in the sample.
 12. The methodof claim 11, wherein the compound which binds to the polypeptide is anantibody.
 13. A kit comprising a compound which selectively binds to apolypeptide of claim 8 and instructions for use.
 14. A method fordetecting the presence of a nucleic acid molecule of claim 1 in a samplecomprising: a) contacting the sample with a nucleic acid probe or primerwhich selectively hybridizes to the nucleic acid molecule; and b)determining whether the nucleic acid probe or primer binds to a nucleicacid molecule in the sample to thereby detect the presence of a nucleicacid molecule of any one of claims 1 in the sample.
 15. The method ofclaim 14, wherein the sample comprises mRNA molecules and is contactedwith a nucleic acid probe.
 16. A kit comprising a compound whichselectively hybridizes to a nucleic acid molecule of claim 1 andinstructions for use.
 17. A method for identifying a compound whichbinds to a polypeptide of claim 8 comprising: a) contacting thepolypeptide, or a cell expressing the polypeptide with a test compound;and b) determining whether the polypeptide binds to the test compound.18. The method of claim 17, wherein the binding of the test compound tothe polypeptide is detected by a method selected from the groupconsisting of: a) detection of binding by direct detection of testcompound/polypeptide binding; b) detection of binding using acompetition binding assay; and c) detection of binding using an assayfor 38594, 57312, 53659, 57250, 63760, 49938, 32146, 57259, 67118,67067, 62092, FBH58295FL, 57255, or 57255alt activity.
 19. A method formodulating the activity of a polypeptide of claim 8 comprisingcontacting the polypeptide or a cell expressing the polypeptide with acompound which binds to the polypeptide in a sufficient concentration tomodulate the activity of the polypeptide.
 20. A method for identifying acompound which modulates the activity of a polypeptide of claim 8comprising: a) contacting a polypeptide of claim 8 with a test compound;and b) determining the effect of the test compound on the activity ofthe polypeptide to thereby identify a compound which modulates theactivity of the polypeptide.